Human serum albumin linkers and conjugates thereof

ABSTRACT

Disclosed is a human serum albumin (HSA) linker and HSA linker with binding, diagnostic, and therapeutic agents conjugated thereto. Also disclosed is a conjugate in which the HSA linker is covalently bonded to amino and carboxy terminal binding moieties that are first and second single-chain Fv molecules (scFvs). Exemplified conjugates are useful, e.g., in reducing tumor cell proliferation, e.g., for therapeutic therapeutic applications. Also disclosed are methods and kits for the diagnostic and therapeutic application of an HSA linker conjugate.

FIELD OF THE INVENTION

Provided are a human serum albumin (HSA) linker conjugates and binding,diagnostic, and therapeutic conjugates thereof. In one embodiment, theHSA linker includes two amino acid substitutions. Another embodiment isa conjugate in which the HSA linker is covalently bonded to amino andcarboxy terminal binding moieties that are first and second single-chainFv molecules (scFvs). Exemplified conjugates are useful, e.g., inreducing tumor cell proliferation, e.g., for therapeutic applications.Further provided are methods for the manufacture and administration ofdiagnostic and therapeutic HSA linker conjugates.

BACKGROUND OF THE INVENTION

Antibody-like binding moieties (including those in intact antibodies,antibody fragments, and scFvs) are often used for therapeuticapplications. Antibody fragments and scFvs generally exhibit shorterserum half lives than intact antibodies, and in some therapeuticapplications increased in vivo half lives would be desirable fortherapeutic agents possessing the functionality of such fragments andscFvs.

Human serum albumin (HSA) is a protein of about 66,500 kD and iscomprised of 585 amino acids including at least 17 disulphide bridges.As with many of the members of the albumin family, human serum albuminplays an important role in human physiology and is located in virtuallyevery human tissue and bodily secretion. HSA has the ability to bind andtransport a wide spectrum of ligands throughout the circulatory system,including the long-chain fatty acids, which are otherwise insoluble incirculating plasma.

The serum albumins belong to a family of proteins that includesalpha-fetoprotein and human group-specific component, also known asvitamin-D binding protein. The serum albumins are the major solubleproteins of the circulatory system and contribute to many vitalphysiological processes. Serum albumin generally comprises about 50% ofthe total blood component by dry weight. The albumins and their relatedblood proteins also play an important role in the transport,distribution, and metabolism of many endogenous and exogenous ligands inthe human body, including a variety of chemically diverse molecules,such as fatty acids, amino acids, steroids, calcium, metals such ascopper and zinc, and various pharmaceutical agents. The albumin familyof molecules is generally thought to facilitate transfer of many ofthese ligands across organ-circulatory interfaces, such as the liver,intestines, kidneys, and the brain. The albumins are thus involved in awide range of circulatory and metabolic functions.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an HSA linker conjugate thatincludes a human serum albumin (HSA) linker that comprises an amino acidsequence set forth in any one of SEQ ID NOS:6-15 and first and secondbinding moieties selected from antibodies, single-chain Fv molecules,bispecific single chain Fv ((scFv′)₂) molecules, domain antibodies,diabodies, triabodies, hormones, Fab fragments, F(ab′)₂ molecules,tandem scFv (taFv) fragments, receptors (e.g., cell surface receptors),ligands, aptamers, and biologically-active fragments thereof, in whichthe first binding moiety is bonded to the amino terminus of the HSAlinker and the second binding moiety is bonded to the carboxy terminusof the HSA linker. In one embodiment, the first binding moietyspecifically binds ErbB3 and the second binding moiety specificallybinds ErbB2. In other embodiments, the HSA linker comprises an aminoacid sequence set forth in SEQ ID NO:1, 9, 10, 14 or 15.

In a second aspect, three or more binding moieties (e.g., 4, 5, 6, 7, 8,9, 10, or more) can be included in the agent; these additional bindingmoieties can be added to the agent, e.g., in tandem (e.g., 2, 3, 4, or 5or more in tandem) with the first or second binding moiety.

In a third aspect, the invention provides an HSA linker that comprisesan amino acid sequence having at least 90% sequence identity to thesequence set forth in SEQ ID NO:1, and a serine residue at position 34and a glutamine residue at position 503 of the amino acid sequence setforth in SEQ ID NO:1. In one embodiment, the amino acid sequence has atleast 95% sequence identity to the sequence set forth in SEQ ID NO:1. Inanother embodiment, the HSA linker comprises the amino acid sequence setforth in SEQ ID NO:1. In yet another embodiment, the HSA linker has theamino acid sequence set forth in SEQ ID NO:1.

In a fourth aspect, the invention provides an HSA linker conjugate thatincludes an HSA linker having at least 90% amino acid sequence identityto the sequence set forth in SEQ ID NO:1 and at least a first bindingmoiety. In one embodiment, the HSA linker conjugate includes a firstpeptide connector that binds the first binding moiety to the HSA linker.

In a fifth aspect, the invention features an HSA linker conjugate thatincludes an HSA linker having an amino acid sequence set forth in anyone of SEQ ID NOs:11-15, or a fragment or variant of any one of thesesequences, and at least a first binding moiety.

In certain embodiments of either the fourth or fifth aspect of theinvention, the HSA linker conjugate further includes a first peptideconnector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) that binds the firstbinding moiety to the amino or carboxy terminus of the HSA linker. In anembodiment, the first connector covalently binds the first bindingmoiety to the HSA linker.

In certain embodiments of either the fourth or fifth aspect of theinvention, the HSA linker conjugate further includes at least a secondbinding moiety. In an embodiment, the HSA linker conjugate furtherincludes a second peptide connector (e.g., AAS, AAQ, or AAAL (SEQ IDNO:5)) that binds the second binding moiety to the HSA linker. In otherembodiments, the second connector binds the second binding moiety to theamino or carboxy terminus of the HSA linker. In a further embodiment,the second connector covalently binds the second binding moiety to theHSA linker. In other embodiments, the HSA linker conjugate furtherincludes three or more binding moieties which are included in tandemwith the first or second binding moiety; the three or more bindingmoieties can further include a connector sequence that joins the threeor more binding moieties to the first or second binding moiety and toeach other.

In certain embodiments of either the fourth or fifth aspect of theinvention, the HSA linker conjugate includes a first peptide connectorthat covalently binds a first binding moiety to the amino terminus ofthe HSA linker and a second peptide connector that covalently binds asecond binding moiety to the carboxy terminus of the HSA linker. In oneembodiment, the first connector has the amino acid sequence AAS or AAQand the second connector has the amino acid sequence set forth in SEQ IDNO:5.

In certain embodiments of either the fourth or fifth aspect of theinvention, the first or second binding moiety (or third or more bindingmoiety) is an antibody, single-chain Fv molecule, bispecific singlechain Fv ((scFv′)₂) molecule, domain antibody, diabody, triabody,hormone, Fab fragment, F(ab′)₂ molecule, tandem scFv (taFv) fragment,receptor (e.g., cell surface receptor), ligand, aptamer, orbiologically-active fragment thereof. In other embodiments, the HSAlinker conjugates provided herein include combinations of thesedifferent types of binding moieties. In one embodiment, at least thefirst or second binding moiety is a human or humanized single-chain Fvmolecule.

In an embodiment of any one of the first, second, third, fourth, orfifth aspects of the invention, one or more of the first or secondbinding moiety (or, if present, the third or further binding moiety) isor specifically binds to a protein selected from the group consisting ofan insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-likegrowth factor (IGF), mesenchymal epithelial transition factor receptor(c-met; also known as hepatocyte growth factor receptor (HGFR)),hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR),epidermal growth factor (EGF), heregulin, fibroblast growth factorreceptor (FGFR), platelet-derived growth factor receptor (PDGFR),platelet-derived growth factor (PDGF), vascular endothelial growthfactor receptor (VEGFR), vascular endothelial growth factor (VEGF),tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha(TNF-α), TNF-β, folate receptor (FOLR), folate, transferrin receptor(TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin(e.g., an α4 integrin or a β-1 integrin), P-selectin,sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate receptor,leukocyte function antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27,CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD106 (vascular celladhesion molecule 1 (VCAM1), CD166 (activated leukocyte cell adhesionmolecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-relatedapoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5,CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin 1 (IL-1),CTLA-4, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressincell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA),Lewis^(Y), MUC-1, epithelial cell adhesion molecule (EpCAM), cancerantigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72antigen, and fragments thereof. In a further embodiment, one or more ofthe first or second binding moiety (or, if present, the third or furtherbinding moiety) is or specifically binds to erythroblastic leukemiaviral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor; ErbB2receptor; ErbB3 receptor; and ErbB4 receptor). In another embodiment,one or more of the first or second binding moiety (or, if present, thethird or further binding moiety) is or specifically binds toalpha-fetoprotein (AFP) or an interferon, or a biologically-activefragment thereof. In a further embodiment, one or more of the first orsecond binding moiety (or, if present, the third or further bindingmoiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab,bevacizumab, daclizumab, efalizumab, golimumab, certolizumab,trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab,or anakinra.

In any one of the first, second, third, fourth, or fifth aspects of theinvention, the HSA linker conjugate is conjoined to a diagnostic, atherapeutic agent, or both. In one embodiment, the diagnostic agent is adetectable label, such as a radioactive, fluorescent, or heavy metallabel. In another embodiment, the therapeutic agent is a cytotoxic,cytostatic, or immunomodulatory agent. Cytotoxic agents includealkylating agents, antibiotics, antineoplastic agents, antiproliferativeagents, antimetabolites, tubulin inhibitors, topoisomerase I or IIinhibitors, hormonal agonists or antagonists, immunomodulators, DNAminor groove binders, and radioactive agents, or any agent capable ofbinding to and killing a tumor cell or inhibiting tumor cellproliferation. Antineoplastic agents include cyclophosphamide,camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin,rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin,caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin,paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene,letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and theirderivatives.

In any one of the first, second, third, fourth, or fifth aspects of theinvention, the HSA linker conjugate is admixed with a pharmaceuticallyacceptable carrier, excipient, or diluent. In one embodiment, the agentexhibits an in vivo half-life of between 6 hours and 7 days. In anotherembodiment, the agent exhibits an in vivo half-life greater than 8hours.

In a sixth aspect, the invention features a method for treating a mammalhaving a disease or disorder by administering any one of the HSA linkerconjugates described herein. In one embodiment, the disease or disorderis associated with cellular signaling through a cell surface receptor.In another embodiment, the mammal is a human. In a further embodiment,the disease or disorder is a proliferative or autoimmune disease.Proliferative diseases include such cancers as melanoma, clear cellsarcoma, head and neck cancer, bladder cancer, breast cancer, coloncancer, ovarian cancer, endometrial cancer, gastric cancer, pancreaticcancer, renal cancer, prostate cancer, salivary gland cancer, lungcancer, liver cancer, skin cancer, and brain cancer. Autoimmune diseasesinclude multiple sclerosis, psoriasis, myasthenia gravis, uveitis,systemic lupus erythematosus, and rheumatoid arthritis. In oneembodiment, the HSA linker conjugate is administered in combination withone or more therapeutic agents, such as an antineoplastic agent.

In a seventh aspect, the invention features a method for making an HSAlinker conjugate by bonding at least a first binding moiety to the aminoterminus and a second binding moiety to the carboxy terminus of an HSAlinker having the amino acid sequence set forth in any one of SEQ IDNOS:1, 3, or 6-15, or a sequence having at least 90%, 95%, 97%, or 100%sequence identity to a sequence set forth in any one of SEQ ID NOS:1, 3,or 6-15. In one embodiment, the first or second binding moiety iscovalently joined to the amino or carboxy terminus of the HSA linker. Inother embodiments, a third or additional binding moiety (e.g., a fourth,fifth, sixth, seventh, eighth, ninth, or tenth binding moiety) iscovalently joined in tandem with the first or second binding moiety tothe amino or carboxy terminus of the HSA linker. In another embodiment,one or more of the first or second binding moiety (or, if present, thethird or further binding moiety) is an antibody, single-chain Fvmolecule, bispecific single chain Fv ((scFv′)₂) molecule, domainantibody, diabody, triabody, hormone, Fab fragment, F(ab′)₂ molecule,tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor),ligand, or aptamer. In another embodiment, the first or second bindingmoiety (or, if present, the third or further binding moiety) is a humanor humanized single-chain Fv molecule. In yet another embodiment, one ormore of the first or second binding moiety (or, if present, the third orfurther binding moiety) is or specifically binds to insulin-like growthfactor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF),mesenchymal epithelial transition factor receptor (c-met; also known ashepatocyte growth factor receptor (HGFR)), hepatocyte growth factor(HGF), epidermal growth factor receptor (EGFR), epidermal growth factor(EGF), heregulin, fibroblast growth factor receptor (FGFR),platelet-derived growth factor receptor (PDGFR), platelet-derived growthfactor (PDGF), vascular endothelial growth factor receptor (VEGFR),vascular endothelial growth factor (VEGF), tumor necrosis factorreceptor (TNFR), tumor necrosis factor alpha (TNF-α), TNF-β, folatereceptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fcreceptor, c-kit receptor, c-kit, an integrin (e.g., an α4 integrin or aβ-1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR),hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD4, CD11,CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor),CD106 (vascular cell adhesion molecule 1 (VCAM1), CD166 (activatedleukocyte cell adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253(TNF-related apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2,CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin1 (IL-1), CTLA-4, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosaladdressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen(CEA), Lewis^(Y), MUC-1, epithelial cell adhesion molecule (EpCAM),cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA),TAG-72 antigen, and fragments thereof. In a further embodiment, one ormore of the first or second binding moiety (or, if present, the third orfurther binding moiety) is or specifically binds to erythroblasticleukemia viral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor;ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In anotherembodiment, one or more of the first or second binding moiety (or, ifpresent, the third or further binding moiety) is or specifically bindsto alpha-fetoprotein (AFP) or an interferon, or a biologically-activefragment thereof. In a further embodiment, one or more of the first orsecond binding moiety (or, if present, the third or further bindingmoiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab,bevacizumab, daclizumab, efalizumab, golimumab, certolizumab,trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab,or anakinra. In another embodiment, the agent is conjoined to adiagnostic or therapeutic agent. In one embodiment, the diagnostic agentis a detectable label, such as a radioactive, bioluminescent,fluorescent, heavy metal, or epitope tag. In another embodiment, thetherapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatoryagent. Cytotoxic agents include alkylating agents, antibiotics,antineoplastic agents, antiproliferative agents, antimetabolites,tubulin inhibitors, topoisomerase I and II inhibitors, hormonal agonistsor antagonists, immunomodulators, DNA minor groove binders, andradioactive agents, or any agent capable of binding to and killing atumor cell or inhibiting tumor cell proliferation. Antineoplastic agentsinclude cyclophosphamide, camptothecin, homocamptothecin, colchicine,combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3,maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil(5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin,tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab,trastuzumab, and their derivatives. In a further embodiment, the agentis admixed with a pharmaceutically acceptable carrier, excipient, ordiluent.

In a eighth aspect, the invention features a method for making an HSAlinker by substituting one or more surface-exposed amino acid residuesin the amino acid sequences set forth in any one of SEQ ID NOS:1, 3, and6-15 with a substitute amino acid capable of chemical modification thatallows conjugation of a diagnostic or therapeutic agent. In oneembodiment, the substitute amino acid is cysteine and the surfaceexposed amino acid residues are serine or threonine. In anotherembodiment, the chemical modification results in a covalent bond betweenthe substitute amino acid and the diagnostic or therapeutic agent. In afurther embodiment, the surface-exposed amino acid residues is threonineat position 496, serine at position 58, threonine at position 76,threonine at position 79, threonine at position 83, threonine atposition 125, threonine at position 236, serine at position 270, serineat position 273, serine at position 304, serine at position 435,threonine at position 478, threonine at position 506, or threonine atposition 508.

In a ninth aspect, the invention features a method for making an HSAlinker by substituting one or more of the residues in the amino acidsequences set forth in any one of SEQ ID NOS:1, 3, and 6-15 with anasparagine, serine, or threonine, thereby incorporating a glycosylationsite within the HSA agent.

In a tenth aspect, the invention features a method for making an HSAlinker by substituting one or more of the asparagine, serine, orthreonine residues in the amino acid sequences set forth in any one ofSEQ ID NOS:1, 3, and 6-15 with any amino acid other than asparagine,serine, or threonine, thereby removing a glycosylation site from the HSAagent.

In an eleventh aspect, the invention features an HSA linker thatcomprises a sequence that has at least 90% sequence identity to one ofthe amino acid sequences set forth in SEQ ID NOS:16-25. In oneembodiment, the HSA linker has at least 95% sequence identity to one ofthe amino acid sequences set forth in SEQ ID NOS:16-25. In anotherembodiment, the HSA linker comprises one of the amino acid sequences setforth in SEQ ID NOS:16-25. In yet another embodiment, the HSA linker hasone of the amino acid sequences set forth in SEQ ID NOS:16-25.

In a further embodiment, the HSA linker or HSA linker conjugate isconjoined to a diagnostic or therapeutic agent. Diagnostic agentsinclude detectable labels, such as a radioactive, bioluminescent,fluorescent, or heavy metal labels, or epitope tags. Fluorescentmolecules that can serve as detectable labels include green fluorescentprotein (GFP), enhanced GFP (eGFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), red fluorescent protein (RFP), anddsRed. In one embodiment, the bioluminescent molecule is luciferase. Inanother embodiment, the epitope tag is c-myc, hemagglutinin, or ahistidine tag. In a further embodiment, the therapeutic agent is acytotoxic polypeptide such as cytochrome c, caspase 1-10, granzyme A orB, tumor necrosis factor-alpha (TNF-α), TNF-β, Fas, Fas ligand,Fas-associated death doman-like IL-1β converting enzyme (FLICE),TRAIL/APO2L, TWEAK/APO3L, Bax, Bid, Bik, Bad, Bak, RICK, vascularapoptosis inducing proteins 1 and 2 (VAP1 and VAP2), pierisin,apoptosis-inducing protein (AIP), IL-1α propiece polypeptide, apoptin,apoptin-associated protein 1 (AAP-1), endostatin, angiostatin, andbiologically-active fragments thereof. An HSA linker or HSA linkerconjugate can be combined with (e.g., conjoined to or mixed with in apharmaceutical composition) one or more therapeutic agents such ascyclophosphamide, camptothecin, homocamptothecin, colchicine,combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3,maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil(5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin,tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab,trastuzumab, and derivatives thereof.

In an embodiment of any aspect described herein, the first and secondbinding moieties (and, if present, one or more of the third or furtherbinding moiety) specifically bind the same target molecule. In anotherembodiment of any aspect, the first and second binding moieties (and, ifpresent, one or more of the third or further binding moiety)specifically bind different target molecules. In a further embodiment ofany aspect, the first and second binding moieties (and, if present, oneor more of the third or further binding moiety) specifically binddifferent epitopes on the same target molecule.

In a twelfth aspect, the invention features an HSA linker that comprisesone or both of amino acid residues 25-44 and 494-513 of the amino acidsequence set forth in SEQ ID NO:1. In one embodiment, the HSA linkercomprises amino acid residues 25-70 and 450-513 of the amino acidsequence set forth in SEQ ID NO:1. In another embodiment, the HSA linkercomprises amino acid residues 15-100 and 400-520 of the amino acidsequence set forth in SEQ ID NO:1. In a further embodiment, the HSAlinker comprises amino acid residues 10-200 and 300-575 of the aminoacid sequence set forth in SEQ ID NO:1. In another embodiment, the HSAlinker comprises amino acid residues 5-250 and 275-580 of the amino acidsequence set forth in SEQ ID NO:1.

In the twelfth aspect of the invention, the HSA linker is conjoined toat least a first binding moiety, for form an HSA linker conjugate. Inone embodiment, the HSA linker conjugate includes at least a firstpeptide connector that binds the first binding moiety to the amino orcarboxy terminus of the HSA linker. In another embodiment, the firstpeptide connector covalently binds the first binding moiety to the HSAlinker. In a further embodiment, the HSA linker includes a secondbinding moiety. In one embodiment, the HSA linker includes a secondpeptide connector that binds the second binding moiety to the HSAlinker. In other embodiments, the second connector binds the secondbinding moiety to the amino or carboxy terminus of the HSA linker. In afurther embodiment, the second connector covalently binds the secondbinding moiety to the HSA linker. In other embodiments, the HSA linkerincludes a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenthbinding moiety. In other embodiments, these additional binding moietiesare present in tandem with one or both of the first or second bindingmoiety. In yet other embodiments, a peptide connector (e.g., AAS, AAQ,or AAAL (SEQ ID NO:5)) separates one or more of these additional bindingmoities from each other, the first or second binding moiety, or the HSAlinker.

In the twelfth aspect of the invention, the HSA linker includes a firstpeptide connector that covalently binds a first binding moiety to theamino terminus of the polypeptide linker and a second peptide connectorthat covalently binds a second binding moiety to the carboxy terminus ofthe HSA linker. In one embodiment, the first connector has the aminoacid sequence AAS or AAQ and the second connector has the amino acidsequence set forth in SEQ ID NO:5.

In the twelfth aspect of the invention, one or more of the first orsecond binding moiety (or, if present, the third or further bindingmoiety) is an antibody, single-chain Fv molecule, bispecific singlechain Fv ((scFv′)₂) molecule, domain antibody, diabody, triabody,hormone, Fab fragment, F(ab′)₂ molecule, tandem scFv (taFv) fragment,receptor (e.g., cell surface receptor), ligand, aptamer, orbiologically-active fragment thereof. In one embodiment, one or more ofthe first or second binding moiety (or, if present, the third or furtherbinding moiety) is a human or humanized single-chain Fv molecule.

In the twelfth aspect of the invention, one or more of the first orsecond binding moiety (or, if present, the third or further bindingmoiety) is or specifically binds to a protein selected from the groupconsisting of an insulin-like growth factor 1 receptor (IGF1R), IGF2R,insulin-like growth factor (IGF), mesenchymal epithelial transitionfactor receptor (c-met; also known as hepatocyte growth factor receptor(HGFR)), hepatocyte growth factor (HGF), epidermal growth factorreceptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblastgrowth factor receptor (FGFR), platelet-derived growth factor receptor(PDGFR), platelet-derived growth factor (PDGF), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factor(VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factoralpha (TNF-α), TNF-β, folate receptor (FOLR), folate, transferrinreceptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, anintegrin (e.g., an α4 integrin or a β-1 integrin), P-selectin,sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate receptor,leukocyte function antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27,CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD106 (vascular celladhesion molecule 1 (VCAM1), CD166 (activated leukocyte cell adhesionmolecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-relatedapoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5,CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin 1 (IL-1),CTLA-4, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressincell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA),Lewis^(Y), MUC-1, epithelial cell adhesion molecule (EpCAM), cancerantigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72antigen, and fragments thereof. In a further embodiment, the first orsecond binding moiety is or specifically binds to erythroblasticleukemia viral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor;ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In anotherembodiment, one or more of the first or second binding moiety (or, ifpresent, the third or further binding moiety) is or specifically bindsto alpha-fetoprotein (AFP) or an interferon, or a biologically-activefragment thereof. In a further embodiment, one or more of the first orsecond binding moiety (or, if present, the third or further bindingmoiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab,bevacizumab, daclizumab, efalizumab, golimumab, certolizumab,trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab,or anakinra.

In the twelfth aspect of the invention, the HSA linker is conjoined to adiagnostic agent, a therapeutic agent, or both. In one embodiment, thediagnostic agent is a detectable label, such as a radioactive,fluorescent, or heavy metal label. In another embodiment, thetherapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatoryagent. Cytotoxic agents include alkylating agents, antibiotics,antineoplastic agents, antiproliferative agents, antimetabolites,tubulin inhibitors, topoisomerase I or II inhibitors, hormonal agonistsor antagonists, immunomodulators, DNA minor groove binders, andradioactive agents, or any agent capable of binding to and killing atumor cell or inhibiting tumor cell proliferation. Antineoplastic agentsinclude cyclophosphamide, camptothecin, homocamptothecin, colchicine,combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3,maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil(5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin,tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab,trastuzumab, and their derivatives. In one embodiment, the conjoined HSAlinker is admixed with a pharmaceutically acceptable carrier, excipient,or diluent. In another embodiment, the HSA linker exhibits an in vivohalf-life of between 6 hours and 7 days. In a further embodiment, theHSA linker exhibits an in vivo half-life greater than 8 hours.

A thirteenth aspect of the invention features an agent of any of theprior aspects of the invention (one through twelve), in which the HSAlinker is replaced by another polypeptide linker. For example, thepolypeptide linker sequence could be a mammalian, non-human serumalbumin polypeptide sequence, such as, e.g., a bovine, murine, feline,and canine serum albumin (BSA) polypeptide sequence. In otherembodiments this polypeptide linker sequence is between 5 and 1,000amino acids in length, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, or 900 amino acids in length, or anynumber of amino acids within this range. In other embodiments, thepolypeptide linker sequence includes a single amino acid (including, butnot limited to, e.g., glycine, alanine, serine, glutamine, leucine, andvaline), or combinations of amino acids.

In another embodiment, the HSA linker is replaced by analpha-fetoprotein (AFP) polypeptide, e.g., mammalian AFP polypeptide,such as a human, murine, bovine, or canine AFP polypeptide. In anembodiment, the AFP linker corresponds to the full-length human AFPpolypeptide sequence (a.a. 1-609; SEQ ID NO:58), the mature human AFPpolypeptide sequence lacking amino acids 1-18 of the signal sequence(a.a., 19-609 of SEQ ID NO:58), or fragments thereof. In otherembodiments, the AFP polypeptide linker contains at least 5 to 8contiguous amino acids, preferably at least 10, 20, or 50 contiguousamino acids, more preferably at least 100 contiguous amino acids, andmost preferably at least 200, 300, 400, or more contiguous amino acidsof SEQ ID NO:58, or has at least 90% sequence identity (e.g., at least95%, 97%, 99%, or more sequence identity) to a contiguous polypeptidesequence of SEQ ID NO:58 having one or more of these lengths. Forexample, an AFP polypeptide linker sequence having 90% sequence identityto a 34-mer human AFP peptide corresponding to amino acids 446-479 ofSEQ ID NO:58 (LSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGV; SEQ ID NO:59) maycontain up to 3 amino acids altered from the 446-479 segment of SEQ IDNO:58. One such example of sequence deviation in biologically activehuman AFP fragments is found in, e.g., U.S. Pat. No. 5,707,963(incorporated by reference herein), which discloses a 34-amino acidfragment of human AFP (SEQ ID NO:59) with flexibility at two amino acidresidues (amino acid 9 and 22 of SEQ ID NO:59). Other examples of AFPpolypeptide linker sequences include, e.g., amino acids 19-198 of SEQ IDNO:58 (human AFP Domain I), amino acids 217-408 of SEQ ID NO:58 (humanAFP Domain II), amino acids 409-609 of SEQ ID NO:58 (human AFP DomainIII), amino acids 19-408 of SEQ ID NO:58 (human AFP Domain I+II), aminoacids 217-609 of SEQ ID NO:58 (human AFP Domain II+III), and amino acids285-609 of SEQ ID NO:58 (human AFP Fragment I). In another embodiment,the human AFP polypeptide linker sequence is an 8-amino acid sequencethat includes amino acids 489-496 (i.e., EMTPVNPG) of SEQ ID NO:58.

A fourteenth aspect of the invention features kits that include any ofthe HSA linkers, HSA linker conjugates, or any other agents described inthe first, second, third, fourth, fifth, eleventh, twelfth, andthirteenth aspects discussed above. The kits further includeinstructions to allow a practitioner (e.g., a physician, nurse, orpatient) to administer the compositions and agents contained therein. Inan embodiment, the kits include multiple packages of a single- ormulti-dose pharmaceutical composition containing an effective amount ofan agent, e.g., HSA linker conjugate as described herein or an HSAlinker that includes, e.g., one or more binding moieties (e.g.,antibodies or antibody fragments (e.g., scFv)), diagnostic agents (e.g.,radionuclide or chelating agents), and/or therapeutic agents (e.g.,cytotoxic or immunomodulatory agents). Optionally, instruments ordevices necessary for administering the pharmaceutical composition(s)may be included in the kits. For instance, a kit may provide one or morepre-filled syringes containing an effective amount of an HSA linkerconjugate or HSA linker, or any binding, diagnostic, and/or therapeuticagent conjugated thereto. Furthermore, the kits may also includeadditional components such as instructions or administration schedulesfor a patient suffering from a disease or condition (e.g., a cancer,autoimmune disease, or cardiovascular disease) to use the pharmaceuticalcomposition(s) containing, e.g., an HSA linker conjugate or HSA linker,or any binding, diagnostic, and/or therapeutic agent conjugated thereto.

DEFINITIONS

The term “antibody” as used interchangeably herein, includes wholeantibodies or immunoglobulins and any antigen-binding fragment or singlechains thereof. Antibodies, as used herein, can be mammalian (e.g.,human or mouse), humanized, chimeric, recombinant, syntheticallyproduced, or naturally isolated. In most mammals, including humans,whole antibodies have at least two heavy (H) chains and two light (L)chains connected by disulfide bonds. Each heavy chain consists of aheavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. The heavy chain constant region consists of threedomains, C_(H)1, C_(H)2, and C_(H)3 and a hinge region between C_(H)1and C_(H)2. Each light chain consists of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region consists of one domain, C_(L). The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. Antibodies of thepresent invention include all known forms of antibodies and otherprotein scaffolds with antibody-like properties. For example, theantibody can be a human antibody, a humanized antibody, a bispecificantibody, a chimeric antibody, or a protein scaffold with antibody-likeproperties, such as fibronectin or ankyrin repeats. The antibody alsocan be a Fab, Fab′2, scFv, SMIP, diabody, nanobody, aptamers, or adomain antibody. The antibody can have any of the following isotypes:IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, andIgAsec), IgD, or IgE. Antibodies that can be used as binding moieties,as defined herein, in combination with an HSA linker include, but arenot limited to, natalizumab, infliximab, adalimumab, rituximab,alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab,certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab,and panitumumab.

The term “antibody fragment,” as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., ErbB2). The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include but are not limited to: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L), and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb including V_(H) and V_(L) domains; (vi) a dAbfragment (Ward et al., Nature 341:544-546 (1989)), which consists of aV_(H) domain; (vii) a dAb which consists of a V_(H) or a V_(L) domain;(viii) an isolated complementarity determining region (CDR); and (ix) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al., Science 242:423-426 (1988) and Huston etal., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies. Antibody fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins.

By “autoimmune disease” is meant a disease in which an immune systemresponse is generated against self epitopes or antigens. Examples ofautoimmune diseases include, but are not limited to, alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet'sdisease, bullous pemphigoid, cardiomyopathy, celiac Sprue-dermatitis,chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, discoid lupus, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Grave's disease, Guillain-Barré syndrome,Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulindependent diabetes, juvenile arthritis, lichen planus, lupus, Ménière'sdisease, mixed connective tissue disease, multiple sclerosis, pemphigusvulgaris, pernicious anemia, polyarteritis nodosa, polychondritis,polyglandular syndromes, polymyalgia rheumatica, polymyositis anddermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis,psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever,rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome,Stiff-Man syndrome, systemic lupus erythematosus (SLE), Takayasuarteritis, temporal arteritis/giant cell arteritis, ulcerative colitis,uveitis (e.g., birdshot retinochoroidopathy uveitis and sarcoiduveitis), vasculitis, vitiligo, Wegener's granulomatosis, and myastheniagravis.

By “binding moiety” is meant any molecule that specifically binds to atarget epitope, antigen, ligand, or receptor. Binding moieties includebut are not limited to antibodies (e.g., monoclonal, polyclonal,recombinant, humanized, and chimeric antibodies), antibody fragments(e.g., Fab fragments, Fab′2, scFv antibodies, SMIP, domain antibodies,diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and domainantibodies), receptors, ligands, aptamers, and other molecules having aknown binding partner.

By “biologically-active” is meant that a molecule, including biologicalmolecules, such as nucleic acids, peptides, polypeptides, and proteins,exerts a physical or chemical activity on itself or other molecule. Forexample, a “biologically-active” molecule may possess, e.g., enzymaticactivity, protein binding activity (e.g., antibody interactions), orcytotoxic activities are “biologically-active.”

The term “chimeric antibody” refers to an immunoglobulin or antibodywhose variable regions derive from a first species and whose constantregions derive from a second species. Chimeric antibodies can beconstructed, for example, by genetic engineering, from immunoglobulingene segments belonging to different species (e.g., from a mouse and ahuman).

By “connector” or “peptide connector” is meant an amino acid sequence of2 to 20 residues in length that is covalently attached to one or both ofthe amino or carboxy termini of an HSA linker, or is covalently attachedto one or more residues of an HSA linker (e.g., a residue between theamino and carboxy terminal residues). In preferred embodiments, thepeptide connector attached to the amino terminus of an HSA linker hasthe amino acid sequence AAS or AAQ and the connector attached to thecarboxy terminus has the amino acid sequence “AAAL” (SEQ ID NO:5).

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” means an amount of an agent (e.g., anHSA linker bonded with one or more binding moieties or diagnostic ortherapeutic agents with or without a connector sequence) sufficient toproduce a desired result, for example, killing a cancer cell, reducingtumor cell proliferation, reducing inflammation in a diseased tissue ororgan, or labeling a specific population of cells in a tissue, organ, ororganism (e.g., a human).

The term “human antibody,” as used herein, is intended to includeantibodies, or fragments thereof, having variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences as described, for example, by Kabat et al.,(Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242(1991)). Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences (i.e., a humanized antibodyor antibody fragment).

The term “humanized antibody” refers to any antibody or antibodyfragment that includes at least one immunoglobulin domain having avariable region that includes a variable framework region substantiallyderived from a human immunoglobulin or antibody and complementaritydetermining regions (e.g., at least one CDR) substantially derived froma non-human immunoglobulin or antibody.

As used herein, an “inflammatory signaling inhibitor” or “ISI” is anagent that decreases the binding between a pro-inflammatory cytokine(e.g., TNF-alpha, TNF-beta, or IL-1) and its receptor (e.g., TNFreceptor 1 or 2, or IL-1 receptor, respectively); decreases the bindingof activating molecules to pro-inflammatory cell surface signalingmolecules (e.g., CD20, CD25, CTLA-4, CD80/CD86, or CD28); or decreasesthe downstream activation of, or activity of, intracellular signalingmolecules that are activated following the binding of pro-inflammatorycytokines to their receptors or the binding of activating molecules topro-inflammatory cell surface signaling molecules (e.g., an agent thatdecreases the activation of, or activity of, signaling molecules in thep38 MAPK signaling pathway). The decrease mediated by an ISI may be adecrease in binding between a pro-inflammatory cytokine and itsreceptor, a decrease in binding of an activating molecule to apro-inflammatory cell surface signaling molecule, or a decrease inintracellular signaling which occurs following the binding ofpro-inflammatory cytokines to their receptors or activating molecules topro-inflammatory cell surface signaling molecules. Preferably, such adecrease mediated by an ISI is a decrease of at least about 10%,preferably at least 20%, 30%, 40%, or 50%, more preferably at least 60%,70%, 80%, or 90% (up to 100%). An ISI may act by reducing the amount ofpro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-1) freelyavailable to bind the receptor. For example, an ISI may be a solublepro-inflammatory cytokine receptor protein (e.g., a soluble TNF receptorfusion protein such as etanercept (ENBREL®) or lenercept), or a solublepro-inflammatory cell surface signaling molecule (e.g., a soluble CTLA-4(abatacept)), or an antibody directed against a pro-inflammatorycytokine or a pro-inflammatory cell surface signaling molecule (e.g., ananti-TNF antibody, such as adalimumab, certolizumab, inflixamab, orgolimumab; an anti-CD20 antibody, such as rituximab; or TRU-015 (TrubionPharmaceuticals)). In addition, an ISI may act by disrupting the abilityof the endogenous wild-type pro-inflammatory cytokine or thepro-inflammatory cell surface signaling molecule to bind to its receptor(e.g., TNF receptor 1 or 2, IL-1 receptor—e.g., anakinra, or CD11a—e.g.,efalizumab (RAPTIVA®, Genentech)). Examples of dominant-negativeTNF-alpha variants are XENP345 (a pegylated version of TNF variantA145R/I97T) and Xpro™1595, and further variants disclosed in U.S. PatentApplication Publication Nos. 20030166559 and 20050265962, hereinincorporated by reference. An example of a dominant negative IL-1variant is anakinra (KINERET®), which is a soluble form of IL-1 thatbinds to the IL-1 receptor without activating intracellular signalingpathways. Inflammatory signaling inhibitors, which can be used in thepresent invention, are also small molecules which inhibit or reduce thesignaling pathways downstream of pro-inflammatory cytokine orpro-inflammatory cell surface signaling molecules (e.g., DE 096).Examples of ISIs of this kind include inhibitors of p38 MAP kinase,e.g., 5-amino-2-carbonylthiopene derivatives (as described in WO04/089929, herein incorporated); ARRY-797; BIRB 796 BS,(1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(morpholin-4-yl-ethoxy)-naphtalen-1-yl]-urea);CHR-3620; CNI-1493; FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan);ISIS 101757 (Isis Pharmaceuticals); ML3404; NPC31145; PD169316; PHZ1112;RJW67657,(4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl)-3-butyn-1-ol;SCIO-469; SB202190; SB203580,(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole);SB239063,trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole;SB242235; SD-282; SKF-86002; TAK 715; VX702; and VX745. Furthermore, anISI may interfere with the processing of a pro-inflammatory cytokine(e.g., TNF-alpha and TNF-beta) from its membrane bound form to itssoluble form. Inhibitors of TACE are ISIs of this class. Examples ofinhibitors of TACE include BB-1101, BB-3103, BMS-561392,butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333,DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI 005.Additional examples of ISIs include short peptides derived from the E.coli heat shock proteins engineered for disease-specificimmunomodulatory activity (e.g., dnaJP1).

By “integrin antagonist” is meant any agent that reduces or inhibits thebiological activity of an integrin molecule (e.g., a reduction orinhibition of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, or more relative to the biological activity in the absence of theintegrin antagonist), such as the α4 subunit of an integrin molecule.The agent may act directly or indirectly on the α4 integrin subunit(NCBI Accession No. P13612; Takada et al., EMBO J. 8:1361-1368 (1989))by inhibiting the activity or expression of the α4 integrin subunit, ormay act on a target to which the intact integrin containing an α4subunit binds. For example, an antibody or blocking peptide that bindsto vascular cell adhesion molecule-1 (VCAM-1), thus preventing thebinding of α4β1 integrin to VCAM-1 is considered an integrin antagonistfor purposes of the present invention. Non-limiting exemplary integrinantagonists suitable for use with the present invention may includeproteins, blocking peptides, antibodies, such as natalizumab (TYSABRI®),and small molecule inhibitors. Examples of α4 integrin antagonistsinclude, but are not limited to, natalizumab (Elan/Biogen Idec; see,e.g., U.S. Pat. Nos. 5,840,299; 6,033,665; 6,602,503; 5,168,062;5,385,839; and 5,730,978; incorporated by reference herein), oMEPUPA-V(Biogen; U.S. Pat. No. 6,495,525; incorporated by reference herein),alefacept, CDP-323 (Celltech); firategrast (SB-68399; GlaxoSmithKline);TR-9109 (Pfizer); ISIS-107248 (Antisense Therapeutics); R-1295 (Roche);and TBC-4746 (Schering-Plough). Additional non-limiting examples of α4integrin antagonists include the small molecules described in U.S. Pat.Nos. 5,821,231; 5,869,448; 5,936,065; 6,265,572; 6,288,267; 6,365,619;6,423,728; 6,426,348; 6,458,844; 6,479,666; 6,482,849; 6,596,752;6,667,331; 6,668,527; 6,685,617; 6,903,128; and 7,015,216 (each hereinincorporated by reference); in U.S. Patent Application Publication Nos.2002/0049236; 2003/0004196; 2003/0018016; 2003/0078249; 2003/0083267;2003/0100585; 2004/0039040; 2004/0053907; 2004/0087574; 2004/0102496;2004/0132809; 2004/0229858; 2006/0014966; 2006/0030553; 2006/0166866;2006/0166961; 2006/0241132; 2007/0054909; and 2007/0232601 (each hereinincorporated by reference); in European Patent Nos. EP 0842943; EP0842944; EP 0842945; EP 0903353; and EP 0918059; and in PCT PublicationNos. WO 95/15973; WO 96/06108; WO 96/40781; WO 98/04247; WO 98/04913; WO98/42656; WO 98/53814; WO 98/53817; WO 98/53818; WO 98/54207; WO98/58902; WO 99/06390; WO 99/06431; WO 99/06432; WO 99/06433; WO99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 99/10312; WO99/10313; WO 99/20272; WO 99/23063; WO 99/24398; WO 99/25685; WO99/26615; WO 99/26921; WO 99/26922; WO 99/26923; WO 99/35163; WO99/36393; WO 99/37605; WO 99/37618; WO 99/43642; WO 01/42215; and WO02/28830; all of which are incorporated by reference herein. Additionalexamples of α4 integrin antagonists include the phenylalaninederivatives described in: U.S. Pat. Nos. 6,197,794; 6,229,011;6,329,372; 6,388,084; 6,348,463; 6,362,204; 6,380,387; 6,445,550;6,806,365; 6,835,738; 6,855,706; 6,872,719; 6,878,718; 6,911,451;6,916,933; 7,105,520; 7,153,963; 7,160,874; 7,193,108; 7,250,516; and7,291,645 (each herein incorporated by reference). Additional amino acidderivatives that are α4 integrin antagonists include those described in,e.g., U.S. Patent Application Publication Nos. 2004/0229859 and2006/0211630 (herein incorporated by reference), and PCT PublicationNos. WO 01/36376; WO 01/47868; and WO 01/70670; all of which areincorporated by reference herein. Other examples of α4 integrinantagonists include the peptides, and the peptide and semi-peptidecompounds described in, e.g., PCT Publication Nos. WO 94/15958; WO95/15973; WO 96/00581; WO 96/06108; WO 96/22966 (Leu-Asp-Val tripeptide;Biogen, Inc.); WO 97/02289; WO 97/03094; and WO 97/49731. An additionalexample of an α4 integrin antagonist is the pegylated molecule describedin U.S. Patent Application Publication No. 2007/066533 (hereinincorporated by reference). Examples of antibodies that are α4 integrinantagonists include those described in, e.g., PCT Publication Nos. WO93/13798; WO 93/15764; WO 94/16094; and WO 95/19790. Additional examplesof α4 integrin antagonists are described herein.

By “interferon” is meant a mammalian (e.g., a human) interferon-alpha,-beta, -gamma, or -tau polypeptide, or biologically-active fragmentthereof, e.g., IFN-α (e.g., IFN-α-1a; see U.S. Patent Application No.20070274950, incorporated herein by reference), IFN-α-1b, IFN-α-2a (seePCT Application No. WO 07/044083, herein incorporated by reference), andIFN-α-2b), IFN-β (e.g., described in U.S. Pat. No. 7,238,344,incorporated by reference; IFN-b-1a (AVONEX® and REBIF®), as describedin U.S. Pat. No. 6,962,978, incorporated by reference, and IFN-β-1b(BETASERON®, as described in U.S. Pat. Nos. 4,588,585; 4,959,314;4,737,462; and 4,450,103; incorporated by reference in their entirety),IFN-g, and IFN-t (as described in U.S. Pat. No. 5,738,845 and U.S.Patent Application Publication Nos. 20040247565 and 20070243163;incorporated by reference).

By “HSA linker conjugate” is meant a human serum albumin (HSA) linker incombination with (preferably covalently linked to) one or more bindingmoieties, peptide connectors, diagnostic agents, or therapeutic agents.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Monoclonal antibodies can be prepared using any art recognizedtechnique and those described herein such as, for example, a hybridomamethod, as described by Kohler et al., Nature 256:495 (1975), atransgenic animal (e.g., Lonberg et al., Nature 368(6474):856-859(1994)), recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567), orusing phage, yeast, or synthetic scaffold antibody libraries using thetechniques described in, for example, Clackson et al., Nature352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

By “pharmaceutically acceptable carrier” is meant a carrier which isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and described, for example, inRemington's Pharmaceutical Sciences, (18^(th) edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa.

By “proliferative disease” or “cancer” is meant any conditioncharacterized by abnormal or unregulated cell growth. Examples ofproliferative diseases include, for example, solid tumors such as:sarcomas (e.g., clear cell sarcoma), carcinomas (e.g., renal cellcarcinoma), and lymphomas; tumors of the breast, colon, rectum, lung,oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, bilecyst,bile duct, small intestine, urinary system (including the kidney,bladder, and epithelium of the urinary tract), female genital system(including the uterine neck, uterus, ovary, chorioma, and gestationaltrophoblast), male genital system (including the prostate, seminalvesicle, and testicles), endocrine glands (including the thyroid gland,adrenal gland, and pituitary body), skin (including angioma, melanoma,sarcoma originating from bone or soft tissue, and Kaposi's sarcoma),brain and meninges (including astrocytoma, neuroastrocytoma,spongioblastoma, retinoblastoma, neuroma, neuroblastoma, neurinoma andneuroblastoma), nerves, eyes, hemopoietic system (includingchloroleukemia, plasmacytoma and dermal T lymphoma/leukemia), and immunesystem (including lymphoma, e.g., Hodgkin's lymphoma and non-Hodgkin'slymphoma). An example of a non-solid tumor proliferative disease isleukemia (e.g., acute lymphoblastic leukemia).

The term “recombinant antibody,” refers to an antibody prepared,expressed, created, or isolated by recombinant means, such as (a)antibodies isolated from an animal (e.g., a mouse) that is transgenic ortranschromosomal for immunoglobulin genes (e.g., human immunoglobulingenes) or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialantibody library (e.g., containing human antibody sequences) usingphage, yeast, or synthetic scaffold display, and (d) antibodiesprepared, expressed, created, or isolated by any other means thatinvolve splicing of immunoglobulin gene sequences (e.g., humanimmunoglobulin genes) to other DNA sequences.

By “specifically bind” is meant the preferential association of abinding moiety (e.g., an antibody, antibody fragment, receptor, ligand,or small molecule portion of an agent as described herein) to a targetmolecule (e.g., a secreted target molecule, such as a cytokine,chemokine, hormone, receptor, or ligand) or to a cell or tissue bearingthe target molecule (e.g., a cell surface antigen, such as a receptor orligand) and not to non-target cells or tissues lacking the targetmolecule. It is recognized that a certain degree of non-specificinteraction may occur between a binding moiety and a non-target molecule(present alone or in combination with a cell or tissue). Nevertheless,specific binding may be distinguished as mediated through specificrecognition of the target molecule. Specific binding results in astronger association between the binding moiety (e.g., an antibody) ande.g., cells bearing the target molecule (e.g., an antigen) than betweenthe binding moiety and e.g., cells lacking the target molecule. Specificbinding typically results in greater than 2-fold, preferably greaterthan 5-fold, more preferably greater than 10-fold and most preferablygreater than 100-fold increase in amount of bound binding moiety (perunit time) to e.g., a cell or tissue bearing the target molecule ormarker as compared to a cell or tissue lacking that target molecule ormarker. Binding moieties bind to the target molecule or marker with adissociation constant of e.g., less than 10⁻⁶M, more preferably lessthan 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²M, and most preferablyless than 10⁻¹³M, 10⁻¹⁴M, or 10⁻¹⁵M. Specific binding to a protein undersuch conditions requires a binding moiety that is selected for itsspecificity for that particular protein. A variety of assay formats areappropriate for selecting binding moieties (e.g., antibodies) capable ofspecifically binding to a particular target molecule. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow &Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

By “sequence identity” is meant (in the context of comparing apolynucleotide or polypeptide sequence to a reference sequence) that thepolynucleotide or polypeptide sequence is the same as the referencesequence or has a specified percentage of nucleotides or amino acidresidues that are the same at the corresponding locations within thereference sequence when the two sequences are optimally aligned.

By “surface-exposed amino acid residue” or “surface-exposed” is meant anamino acid residue that is present on the exterior face of the foldedand conformationally-correct tertiary structure of a HSA polypeptide.Such residues can be substituted with e.g., other, chemically-reactive,amino acids (e.g., cysteine) to allow for site-specific conjugation ofdiagnostic or therapeutic agents. Additionally, surface-exposed aminoacid residues can be substituted to allow (e.g., by addition of serine,threonine, or asparagine residues, or glycosylation motifs) or prevent(e.g., by removal of serine, threonine, or asparagine residues, orglycosylation motifs) glycosylation. Surface-exposed amino acid residuesinclude, but are not limited to, threonine at position 496, serine atposition 58, threonine at position 76, threonine at position 79,threonine at position 83, threonine at position 125, threonine atposition 236, serine at position 270, serine at position 273, serine atposition 304, serine at position 435, threonine at position 478,threonine at position 506, and threonine at position 508 (amino acidnumbering is relative to e.g., the sequence of the HSA linker set forthin SEQ ID NO:1). Other surface-exposed residues can be identified by theskilled artisan using the HSA crystal structure (Sugio et al., “Crystalstructure of human serum albumin at 2.5 A resolution,” Protein Eng.12:439-446 (1999)). A “subject” refers to a human patient or a nudemouse xenograft model comprising human tumor cells.

A “target molecule” or “target cell” is meant a molecule (e.g., aprotein, epitope, antigen, receptor, or ligand) or cell to which abinding moiety (e.g., an antibody), or an HSA conjugate that containsone or more binding moieties (e.g., an HSA linker bonded to one or moreantibodies or antibody fragments) can specifically bind. Preferredtarget molecules are exposed on the exterior of a target cell (e.g., acell-surface or secreted protein) but target molecules may alternatelyor also be present in the interior of a target cell.

“Treating” preferably provides a reduction (e.g., by at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even100%) in the progression or severity of a human disease or disorder(e.g., an autoimmune or proliferative disease), or in the progression,severity, or frequency of one or more symptoms of the human disease ordisorder in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration showing the schematic representation of anexemplary HSA linker conjugate. The connector between the amino terminalbinding moiety and the HSA linker has a sequence of alanine, alanine andserine. The connector between the HSA linker and the carboxy terminalbinding moiety has a sequence of alanine, alanine, alanine, leucine (SEQID NO:5).

FIG. 2 is a graph showing that B2B3 variants inhibit HRG-induced pErbB3in ZR75-1 breast cancer cells.

FIG. 3A is a graph showing the inhibition of phosphorylated ErbB3 inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B1D2-2 (A5-HSA-B1D2. Further details regarding this HSAlinker conjugate is set forth below, e.g., in Table 6.

FIG. 3B is a graph showing the inhibition of phosphorylated ErbB3 inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B1D2-1 (H3-HSA-B1D2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 3C is a graph showing the inhibition of phosphorylated ErbB3 inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 3D is a graph showing the inhibition of phosphorylated ErbB3 inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 4A is a graph showing the inhibition of phosphorylated AKT in BT474breast cancer cells following 24 hour pre-treatment with the HSA linkerconjugate B1D2-2 (A5-HSA-B1D2). Further details regarding this HSAlinker conjugate is set forth below, e.g., in Table 6.

FIG. 4B is a graph showing the inhibition of phosphorylated AKT in BT474breast cancer cells following 24 hour pre-treatment with the HSA linkerconjugate B1D2-1 (H3-HSA-B1D2). Further details regarding this HSAlinker conjugate is set forth below, e.g., in Table 6.

FIG. 4C is a graph showing the inhibition of phosphorylated AKT in BT474breast cancer cells following 24 hour pre-treatment with the HSA linkerconjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding this HSAlinker conjugate is set forth below, e.g., in Table 6.

FIG. 4D is a graph showing the inhibition of phosphorylated AKT in BT474breast cancer cells following 24 hour pre-treatment with the HSA linkerconjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding this HSAlinker conjugate is set forth below, e.g., in Table 6.

FIG. 5A is a graph showing that inhibition of phosphorylated ERK inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B1D2-2 (A5-HSA-B1D2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 5B is a graph showing that inhibition of phosphorylated ERK inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B1D2-1 (H3-HSA-B1D2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 5C is a graph showing that inhibition of phosphorylated ERK inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 5D is a graph showing that inhibition of phosphorylated ERK inBT474 breast cancer cells following 24 hour pre-treatment with the HSAlinker conjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding thisHSA linker conjugate is set forth below, e.g., in Table 6.

FIG. 6 is a graph showing that treatment of BT474 breast cancer cellswith B2B3-1 variants causes G1 arrest and a decrease in the number ofcells in S phase.

FIG. 7 is a flow cytometry histogram showing that pre-incubation ofBT-474-M3 cells with 1 μM B2B3-1 substantially blocks binding of HRG.

FIG. 8A is a graph showing that B2B3-1 inhibits ErbB3 phosphorylation inB-T474-M3 cells. Breast cancer cells BT-474-M3 were pre-treated with adose titration of B2B3-1 for 24 hours and then stimulated for 10 minuteswith 5 nM of HRG 1βEGF domain. The phosphorylation status of ErbB3 wasthen examined using an ELISA assay.

FIG. 8B is a graph showing that B2B3-1 inhibits ErbB3 phosphorylation inZR75-3 0 cells. Breast cancer cells ZR75-3 0 (FIGS. 8B and 8D) werepre-treated with a dose titration of B2B3-1 for 24 hours and thenstimulated for 10 minutes with 5 nM of HRG 1β EGF domain. Thephosphorylation status of ErbB3 was then examined using an ELISA assay.

FIG. 8C is a graph showing that B2B3-1 inhibits AKT phosphorylation inB-T474-M3 cells. Breast cancer cells BT-474-M3 were pre-treated with adose titration of B2B3-1 for 24 hours and then stimulated for 10 minuteswith 5 nM of HRG 1βEGF domain. The phosphorylation status of AKT wasthen examined using an ELISA assay.

FIG. 8D is a graph showing that B2B3-1 inhibits AKT phosphorylation inZR75-3 0 cells. Breast cancer cells ZR75-3 0 were pre-treated with adose titration of B2B3-1 for 24 hours and then stimulated for 10 minuteswith 5 nM of HRG 1βEGF domain. The phosphorylation status of AKT wasthen examined using an ELISA assay.

FIG. 9 is a photograph of a Western blot that shows the effect oftreatment with increasing concentrations of B2B3-1 on signaling proteinsin BT474 breast cancer cells. “p-” indicates the tyrosine-posphorylatedform of the signaling protein. Beta tubulin (not a signaling protein inthis context) provides a loading control. Beta tubulin (not a signalingprotein in this context) provides a loading control.

FIG. 10 is a photograph of a Western blot that shows theimmunoprecipitation of B2B3-1 treated BT474 breast cancer cells. Betatubulin provides a control for levels of cellular proteins input intothe immunoprecipitation reactions.

FIG. 11A is a graph showing B2B3-1 treatment of BT-474 cell line causesG1 arrest and a decrease in the population of cells in S phase (FIG.11A).

FIG. 11B is a graph showing B2B3-1 treatment of BT-474 cell lineinhibits colony formation in both BT-474 and SKBr3 cells compared tountreated cells.

FIG. 11C is a graph showing B2B3-1 inhibits proliferation of BT-474-M3cells in a cell impedance assay.

FIG. 12 is a graph showing that B2B3-1 does not stimulate ErbB3phosphorylation in ZR75-1 cells.

FIG. 13A is a graph showing that B2B3-1 binds specifically to ErbB3.

FIG. 13B is a graph showing that B2B3-1 binds specifically to ErbB2.

FIG. 14 is a graph showing that avidity binding of B2B3-1 to MALME-3cells results in a significant increase in apparent binding affinitycompared to ErbB2-only binding variant, SKO-3, and ErbB3-only bindingvariant, SKO-2.

FIG. 15A is a graph showing the stability of B2B3-1 in mouse serum. 100nM B2B3-1 was incubated in mouse serum at 37° C. for a period of 120hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and theability of B2B3-1 to bind both ErbB2 and ErbB3 was measured by ELISA(RLU=relative light units).

FIG. 15B is a graph showing the stability of B2B3-1 in Cynomolgus monkeyserum. 100 nM B2B3-1 was incubated in Cynomolgus monkey serum at 37° C.for a period of 120 hours. Samples were removed at 0, 24, 48, 72, 96 and120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 wasmeasured by ELISA (RLU=relative light units).

FIG. 15C is a graph showing the stability of B2B3-1 in human serum. 100nM B2B3-1 was incubated in human serum at 37° C. for a period of 120hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and theability of B2B3-1 to bind both ErbB2 and ErbB3 was measured by ELISA(RLU=relative light units).

FIG. 16 is a graph showing B2B3-1 dose response in a BT-474-M3 breastcancer xenograft model. The relationship of B2B3-1 dose on tumorresponse was assessed in the BT-474-M3 breast tumor line at the dosesindicated. HSA was given at 52.5 mg/kg, which is an equimolar dose tothe 90 mg/kg B2B3-1 dose.

FIG. 17A is a graph showing that B2B3-1 is effective in a Calu-3 (humanlung adenocarcinoma) xenograft model in an ErbB2 dependent manner. Micewere treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at anequimolar dose to B2B3-1.

FIG. 17B is a graph showing that B2B3-1 is effective in a SKOV-3 (humanovarian adenocarcinoma) xenograft model in an ErbB2 dependent manner.Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control atan equimolar dose to B2B3-1.

FIG. 17C is a graph showing that B2B3-1 is effective in a NCI-N87 (humangastric carcinoma) xenograft model in an ErbB2 dependent manner. Micewere treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at anequimolar dose to B2B3-1.

FIG. 17D is a graph showing that B2B3-1 is effective in a ACHN (humankidney adenocarcinoma) xenograft model in an ErbB2 dependent manner.Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control atan equimolar dose to B2B3-1.

FIG. 17E is a graph showing that B2B3-1 is effective in a MDA-MB-361(human breast adenocarcinoma) xenograft model in an ErbB2 dependentmanner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSAcontrol at an equimolar dose to B2B3-1.

FIG. 18A is a graph showing that the over-expression of ErbB2 convertsB2B3-1 non-responder ADRr breast cancer xenograft model into aresponder. ErbB2 was over-expressed in wild type ADRr xenografts using aretroviral expression system.

FIG. 18B is a graph showing that the over-expression of ErbB2 convertsB2B3-1 non-responder ADRr breast cancer xenograft model into aresponder. ErbB2 was over-expressed in ADRr-E2 xenografts using aretroviral expression system.

FIG. 19A is a graph showing that B2B3-1 activity correlates positivelywith ErbB2 expression levels in vitro.

FIG. 19B is a graph showing that B2B3-1 activity correlates positivelywith ErbB2 expression levels in vivo.

FIG. 20A shows that B2B3-1 treatment modifies tumor cell cycling. FIG.20A includes fluorescent micrographs showing that B2B3-1 treatment ofBT474-M3 breast tumor cells for 6 hours results in translocation of cellcycle inhibitor p27^(kip1) to the nucleus. Hoechst stain was used toidentify the nucleus.

FIG. 20B is a Western blot of BT-474-M3 cells treated with B2B3-1 for 72hours, which resulted in a decrease in the levels of the cell cycleregulator Cyclin D1. The cytoskeleton protein vinculin was probed as aprotein loading control in this experiment.

FIG. 21A is a micrograph showing that B2B3-1 treatment of BT474 breasttumor xenografts results in translocation of p27^(kip1) to the nucleus.BT474 breast tumor xenografts were treated with B2B3-1 at a dose of 30mg/kg every 3 days for a total of 4 doses and stained for p27^(kip1).

FIG. 21B is a micrograph showing the effect of an equimolar dose of HSAon BT474 breast tumor xenografts treated every 3 days for a total of 4doses and stained for p27^(kip1).

FIG. 22A is a fluorescent micrograph showing that B2B3-1 treatmentresults in a reduction of the proliferation marker Ki67 in BT474-M3breast cancer xenograft. BT474-M3 breast tumor xenografts were treatedwith B2B3-1 at a dose of 30 mg/kg every 3 days for a total of 4 doses.

FIG. 22B is a fluorescent micrograph showing the effect of an equimolardose of HSA on BT474-M3 breast cancer xenograft treated every 3 days fora total of 4 doses.

FIG. 23A is a fluorescent micrograph showing that B2B3-1 treatmentresults in a reduction of vessel density in BT474-M3 breast cancerxenograft tumors (as determined by a reduction in CD31 staining).BT474-M3 breast tumor xenografts were treated with B2B3-1 at a dose of30 mg/kg every 3 days for a total of 4 doses.

FIG. 23B is a fluorescent micrograph showing the effect of an equimolardose of HSA on vessel density in BT474-M3 breast cancer xenograft tumors(as determined by a reduction in CD31 staining) treated every 3 days fora total of 4 doses.

FIG. 24A is a graph showing that B2B3-1 inhibits phosphorylation ofErbB3 in vivo. Lysates from individual BT-474-M3 xenograft tumorstreated with B2B3-1 (M1-M5) or control HSA (H1-H2) were subjected toSDS-PAGE and probed for pErbB3 and beta tubulin using Western blotanalysis.

FIG. 24B is a graph showing that normalization of the mean pErbB3 signalto the mean beta tubulin signal demonstrated that B2B3-1 treated tumorscontained significantly less pErbB3 than HSA tumors.

FIGS. 25A and B are graphs showing the in vivo activity of B2B3-1 inBT-474-M3 shPTEN and shControl xenografts. Cultured BT-474-M3 tumorcells were transfected with a control vector (FIG. 25A) or with aretroviral vector expressing shPTEN (FIG. 25B), which knocks out PTENactivity. BT-474-M3 breast cancer cells thus engineered to knock outPTEN activity were injected into the right flank of mice, while cellstransfected with control vector were injected into the left flank of thesame mouse. Mice were treated with 30 mg/kg B2B3-1 every 3 days or 10mg/kg Herceptin every week and HSA was injected as a control at anequimolar dose to B2B3-1. B2B3-1 and Herceptin promoted a reduction inthe size of tumors formed by control BT-474-M3 breast cancer cells (FIG.25A), whereas only B2B3-1 (and not Herceptin) promoted a reduction inthe size of tumors formed by BT-474-M3 breast cancer cells lackingexpression of PTEN (FIG. 25B).

FIGS. 26A-B show that B2B3-1 inhibits phosphorylation of AKT inBT-474-M3 xenografts that have reduced PTEN activity. Tumors were lysedfollowing the completion of treatment (q3d×11) and tested for PTEN,pErbB3, and pAKT expression levels by Western blot analysis (FIG. 26A).Densitometry on the band intensity for pAKT normalized to total AKT andtotal protein demonstrate that B2B3-1 was able to inhibitphosphorylation of this protein, when Herceptin did not (FIG. 26B).

FIG. 27A is a graph showing the single dose pharmacokinetic propertiesof 5 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serumconcentrations are comparable measured by the HSA assay or ErbB2/ErbB3assay, indicating that the antigen binding activity of B2B3-1 isretained in circulation.

FIG. 27B is a graph showing the single dose pharmacokinetic propertiesof 15 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serumconcentrations are comparable measured by the HSA assay or ErbB2/ErbB3assay, indicating that the antigen binding activity of B2B3-1 isretained in circulation.

FIG. 27C is a graph showing the single dose pharmacokinetic propertiesof 30 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serumconcentrations are comparable measured by the HSA assay or ErbB2/ErbB3assay, indicating that the antigen binding activity of B2B3-1 isretained in circulation.

FIG. 27D is a graph showing the single dose pharmacokinetic propertiesof 45 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serumconcentrations are comparable measured by the HSA assay or ErbB2/ErbB3assay, indicating that the antigen binding activity of B2B3-1 isretained in circulation.

FIG. 28 is a graph showing the dose-exposure relationship for 5, 15, 30,and 45 mg/kg bolus doses of B2B3-1 in nude mice. Increases in doseresult in a linear increase in overall exposure to B2B3-1.

FIG. 29 is a graph showing the B2B3-1 serum concentrations measured inCynomolgus monkeys dosed every three days for 4 doses with 4 mg/kg(n=2), 20 mg/kg (n=2) and 200 mg/kg (up to 336 hour n=4, for 384, 552and 672 hour time points n=2).

FIG. 30 is an illustration of the B2B3-1 expression plasmidpMP10k4H3-mHSA-B1D2.

FIG. 31 is an illustration of the neomycin resistance plasmid pSV2-neo.

FIG. 32 is an illustration of the hygromycin resistance plasmid pTK-Hyg.

FIG. 33 shows data demonstrating that B2B3-1 dosed q7d shows equivalentefficacy to q3d dosing.

FIG. 34 shows western blot data demonstrating that B2B3-1 andtrastuzumab exhibit different mechanisms of ErbB3 inhibition.

FIG. 35 A-C shows the results of the experiments detailed in Example 43in which B2B3-1 combination treatment with trastuzumab was studied inspheroids of various human breast cancer cell lines, which serve as amodel for human breast tumors.

FIG. 35A shows data obtained using BT-474-M3 cells, FIG. 35B shows dataobtained using SKBR3 cells, and FIG. 35C shows data obtained usingMDA-MB-361 cells. The molar concentration of B2B3-1, alone or incombination, is given along the X-axis. The molar concentration oftrastuzumab, alone or in combination is one third that of each indicatedconcentration of B2B3-1.

FIG. 36 show the results of the in vivo tumor xenograft experimentsdetailed in Example 44. “Days” on the X-axis indicates days post tumorimplant. Error bars for each data point represent the response for atleast two independent xenografts.

FIG. 37 shows data obtained from a xenograft model essentially asdescribed in Example 44, except that the tumor cells used are N-87gastric tumor cells which may be obtained from the US National CancerInstitute.

FIG. 38 shows a subset of the data presented in FIG. 36.

DETAILED DESCRIPTION

This invention provides human serum albumin (HSA) linkers, as well asHSA linker conjugates (e.g., binding, diagnostic, or therapeutic agents)that comprise an HSA linker and one or more additional moieties such asbinding moieties. Such HSA linker conjugates have desirable propertiessuch as, for example, an increased in vivo half-life of between 6 hoursand 7 days, and do not induce significant humoral or cell-mediatedimmune responses when administered in vivo to a mammal (e.g., a human).In one aspect, the invention provides a mutated HSA linker that has twodefined amino acid substitutions (i.e., the “C34S” and “N503Q”substitutions, as set forth in SEQ ID NO:1). In another aspect, theinvention provides an HSA linker bonded to one or more binding moieties(e.g., antibodies, antibody fragments, receptor/ligands, or smallmolecules) for diagnostic or therapeutic applications in a mammal (e.g.,a human) in vivo or for use in vitro in connection with mammalian cells,tissues, or organs. In a further aspect, the HSA linker may be coupledto one or more immunomodulatory agents, cytotoxic or cytostatic agents,detectable labels, or radioactive agents for diagnostic or therapeuticapplications in a mammal (or in connection with a mammalian cell,tissue, or organ). An HSA linker conjugate, which includes the HSAlinker, can be optionally combined with one or more pharmaceuticallyacceptable carriers or excipients and can be formulated to beadministered intravenously, intramuscularly, orally, by inhalation,parenterally, intraperitoneally, intraarterially, transdermally,sublingually, nasally, through use of suppositories, transbuccally,liposomally, adiposally, opthalmically, intraocularly, subcutaneously,intrathecally, topically, or locally. An HSA linker conjugate can, butneed not, be combined or coadministered with one or morebiologically-active agents (e.g., biological or chemical agents, such aschemotherapeutics and antineoplastic agents). In a further aspect, theinvention provides a kit, with instructions, for the conjugation ofbinding moieties (e.g., antibodies, antibody fragments, receptors orligands), immunomodulatory agents, cytotoxic or cytostatic agents,detectable labels, or radioactive agents to the HSA linker to prepareHSA linker conjugates that can be used for diagnostic or therapeuticapplications.

Human Serum Albumin (HSA) Linkers

An HSA linker may comprise a wild-type HSA amino acid sequence, as setforth in SEQ ID NO:3. Alternatively, the HSA linker may comprise analtered, or mutated, sequence. One mutated HSA linker contains two aminoacid mutations, at positions 34 and 503, relative to the wild-type HSAamino acid sequence set forth in SEQ ID NO:3. The cysteine residue atposition 34 (i.e., C34) can be mutated to any amino acid residue otherthan cysteine (e.g., serine, threonine, or alanine). Likewise, theasparagine residue at position 503 (i.e., N503) can be mutated to anyamino acid residue other than asparagine (e.g., glutamine, serine,histidine, or alanine). In one embodiment, the HSA linker has the theamino acid and corresponding nucleotide sequence set forth in SEQ IDNOS:1 and 2, respectively. This mutated HSA linker contains two aminoacid substitutions (i.e., serine for cysteine at amino acid residue 34(“C34S”) and glutamine for asparagine at amino acid residue 503(“N503Q”)). The HSA linker, when bonded to one or more binding moieties(e.g., antibodies, antibody fragments (e.g., single chain antibodies),or other targeting or biologically active agents (e.g., receptors andligands)), confers several beneficial pharmacologic properties to thoseconjugates and to additional diagnostic or therapeutic agents alsoconjoined (e.g., immunomodulatory agents, cytotoxic or cytostaticagents, detectable labels, or radioactive agents)) relative to thepharmacologic properties of these agents in the absence of the HSAlinker. These benefits can include decreased immunogenicity (e.g.,decreased host antibody neutralization of linker-antibody conjugates),increased detection of HSA linker conjugates (e.g., by massspectroscopy) and increased pharmacologic half-life (e.g., a half-lifegreater than 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3days, 4 days, 5 days, 6 days, or 7 days) when administered to a mammal(e.g., a human). Specifically, the substitution of serine for cysteineat amino acid residue 34 results in reduced oxidation and proteinheterogeneity of the HSA linker. In wild-type HSA, the asparagine atamino acid residue 503 is sensitive to deamination, likely resulting inreduced pharmacologic half-life. The substitution of glutamine forasparagine at amino acid residue 503 can result in increasedpharmacologic half-life of the HSA linker, and correspondingly, ofconjugate agents that include the HSA linker when administered to amammal (e.g., a human) or cells, tissues, or organs thereof.

In other embodiments, the mutated HSA linker includes domain I of HSA(SEQ ID NO:53; residues 1-197 of SEQ ID NO:1), domain III of HSA (SEQ IDNO:55; residues 381-585 of SEQ ID NO:1), combination of domains I andIII of HSA, or a combination of domain I or III of HSA with domain II ofHSA (SEQ ID NO:54; residues 189-385 of SEQ ID NO:1). For example, an HSAlinker can include domains I and II, I and III, or II and III. Inaddition, the cysteine residue at position 34 (i.e., C34) of domain I(SEQ ID NO:53) can be mutated to any amino acid residue other thancysteine (e.g., serine, threonine, or alanine). Likewise, the asparagineresidue at position 503 (i.e., N503) of domain III (SEQ ID NO:55) can bemutated to any amino acid residue other than asparagine (e.g.,glutamine, serine, histidine, or alanine). These HSA linkers can beincorporated into an HSA linker conjugate, which includes one or more ofa peptide connector, a binding moiety, and therapeutic or diagnosticagents, each of which is described in detail below.

Peptide Connectors

To facilitate the conjugation of binding moieties, as defined herein, tothe HSA linker, short (e.g., 2-20 amino acids in length) peptideconnectors that can be bonded (e.g, covalently (e.g., a peptidic bond),ionically, or hydrophobically bonded, or via a high-affinityprotein-protein binding interaction (e.g., biotin and avidin)) to theamino or carboxy termini of an HSA linker. These connectors provideflexible tethers to which any of the binding moieties described hereincan be attached. A peptide connector may be 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length. Inone embodiment, the connector is a sequence of, e.g., glycine, alanine,serine, glutamine, leucine, or valine residues. Although notspecifically enumerated herein, the connector can be solely glycine,alanine, serine, glutamine, leucine, or valine residues, or it may beany combination of these residues up to about 20 amino acids in length.In a preferred embodiment, the connector attached to the amino terminusof an HSA linker has the amino acid sequence AAS or AAQ and theconnector attached to the carboxy terminus has the amino acid sequence“AAAL” (SEQ ID NO:5). The connector can be covalently bound to the aminoor carboxy terminal residue of the HSA linker, to an amino acid residuewithin the HSA linker, or can be included between one or more bindingmoieties, if present.

HSA Linker Manufacture

The HSA linker, with or without one or more peptide connectors describedabove, one or more of the binding moieties described below,polypeptide-based detectable labels, and other polypeptide-basedtherapeutic agents, can be produced recombinantly. For example, anucleotide sequence encoding the HSA linker (and one or more of theoptional elements) may be expressed (e.g., in a plasmid, viral vector,or transgenically) in a bacterial (e.g., E. coli), insect, yeast, ormammalian cell (e.g., a CHO cell), or a mammalian tissue, organ, ororganism (e.g., a transgenic rodent, ungulate (e.g., a goat), ornon-human primate). After expression of the HSA linker in the host cell,tissue, or organ, the skilled artisan may isolate and purify the HSAlinker using standard protein purification methods (e.g., FPLC oraffinity chromatography). A recombinant expression system for theproduction of an HSA linker in combination with two binding moieties isillustrated in FIG. 1.

Alternatively, the HSA linker, with or without one or more of theoptional elements described above, can be synthetically produced. Forexample, the HSA linker or HSA linker conjugate can be prepared bytechniques generally established in the art of peptide synthesis, suchas the solid-phase approach. Solid-phase synthesis involves the stepwiseaddition of amino acid residues to a growing peptide chain that islinked to an insoluble support or matrix, such as polystyrene. TheC-terminus residue of the peptide is first anchored to a commerciallyavailable support with its amino group protected with an N-protectingagent such as a t-butyloxycarbonyl group (tBoc) or afluorenylmethoxycarbonyl (FMOC) group. The amino-protecting group isremoved with suitable deprotecting agents such as TFA in the case oftBOC or piperidine for FMOC and the next amino acid residue (inN-protected form) is added with a coupling agent such asdicyclocarbodiimide (DCC). Upon formation of a peptide bond, thereagents are washed from the support. After addition of the finalresidue, the agent is cleaved from the support with a suitable reagent,such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF). Ifdesired, the HSA linker, with or without one or more of the optionalelements described above, can be manufactured in one, two, three, ormore segments, which can then be ligated to form the whole HSA linkerconstruct.

Binding Moieties

HSA linker conjugates may include one or more binding moieties, such asantibodies, antibody fragments (as defined herein, e.g., a single chainFv (scFv)) or receptor/ligands (i.e., protein or glycoprotein ligands orreceptors)) that allow selective and specific binding of the HSA linkerconjugate to a target cell, tissue, or organ. The binding moieties canbe bonded to the HSA linker (e.g., via a covalent (e.g., a peptidebond), ionic, or hydrophobic bond, or via a high-affinityprotein-protein binding interaction (e.g., biotin and avidin)).

One or more binding moieties can be bonded to an HSA linker. In oneembodiment, two or more identical binding moieties (i.e., moietieshaving the same structure and binding affinities) are bonded to an HSAlinker, one or more (e.g., in tandem) each at the amino and carboxytermini, the HSA linker thereby affording improved avidity of thebinding moieties for their target antigen. Alternatively, two or moredifferent binding moieties (e.g., an antibody, such as a scFv, withbinding affinities for two or more different target molecules, or scFvwith binding affinities for two or more different epitopes on the sametarget molecule) can be bonded to an HSA linker (e.g., a bispecific HSAlinker conjugate) to allow multiple target antigens or epitopes to bebound by the HSA linker conjugate. In another embodiment, differentspecies of binding moieties can also be bonded to an HSA linker tobestow, for example, two or more different binding specificities oragonistic/antagonistic biological properties on the linker conjugate.Useful combinations of binding moiety pairs for use in the preparationof bispecific HSA linker conjugates are disclosed in, e.g.,International Patent Application Publications WO 2006/091209 and WO2005/117973, herein incorporated by reference. In other embodiments,more than two binding moieties (e.g., the same or different bindingmoieties) can be bonded to an HSA linker to form an HSA linkerconjugate.

The invention features an HSA linker conjugate having at least first andsecond binding moieties, each of which can be bound at either the aminoor carboxy terminus of the HSA linker, or to peptide connectors, asdefined herein, present at either or both termini FIG. 1 illustrates anexemplary mutated HSA linker in which two binding moieties (“arm 1” and“arm 2”) are bonded to the mutated HSA linker by the amino terminalpeptide connector AAS and carboxy terminal peptide connector AAAL (SEQID NO:5). Binding moieties (e.g., an antibody or scFv) can also be boundto other loci (e.g., internal amino acid residues of the HSA linker),for example, covalently or ionically, e.g., using biotin-avidininteractions. Biotinylation of amine (e.g., lysine residues) andsulfhydryl (e.g., cysteine residues) amino acid side chains is known inthe art and can be used to attach binding moieties to the HSA linker.

Binding moieties that can be included in an HSA linker conjugate includeantibodies, antibody fragments, receptors, and ligands. Binding moietiesbound to an HSA linker may be recombinant (e.g., human, murine,chimeric, or humanized), synthetic, or natural. Representative bindingmoieties include, for example, complete antibodies, domain antibodies,diabodies, triabodies, bi-specific antibodies, antibody fragments, Fabfragments, F(ab′)2 molecules, single chain Fv (scFv) molecules,bispecific single chain Fv ((scFv′)₂) molecules, tandem scFv fragments,antibody fusion proteins, hormones, receptors, ligands, and aptamers,and biologically-active fragments thereof.

Antibodies

Antibodies include the IgG, IgA, IgM, IgD, and IgE isotypes. Antibodiesor antibody fragments thereof, as used herein, contain one or morecomplementarity determining regions (CDR) or binding peptides that bindto target proteins, glycoproteins, or epitopes present on the exterioror in the interior of target cells.

Many of the antibodies, or fragments thereof, described herein canundergo non-critical amino-acid substitutions, additions or deletions inboth the variable and constant regions without loss of bindingspecificity or effector functions, or intolerable reduction of bindingaffinity (e.g., below about 10⁻⁷ M). Usually, an antibody or antibodyfragment incorporating such alterations exhibits substantial sequenceidentity to a reference antibody or antibody fragment from which it isderived. Occasionally, a mutated antibody or antibody fragment can beselected having the same specificity and increased affinity comparedwith a reference antibody or antibody fragment from which it wasderived. Phage-display technology offers powerful techniques forselecting such antibodies. See, e.g., Dower et al., WO 91/17271McCafferty et al., WO 92/01047; and Huse, WO 92/06204, incorporated byreference herein.

The HSA linker can also be bonded to one or more fragments of anantibody that retain the ability to bind with specificity to a targetantigen. Antibody fragments include separate variable heavy chains,variable light chains, Fab, Fab′, F(ab′)₂, Fabc, and scFv. Fragments canbe produced by enzymatic or chemical separation of intactimmunoglobulins. For example, a F(ab′)₂ fragment can be obtained from anIgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 usingstandard methods such as those described in Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988). Fabfragments may be obtained from F(ab′)₂ fragments by limited reduction,or from whole antibody by digestion with papain in the presence ofreducing agents. Fragments can also be produced by recombinant DNAtechniques. Segments of nucleic acids encoding selected fragments areproduced by digestion of full-length coding sequences with restrictionenzymes, or by de novo synthesis. Often fragments are expressed in theform of phage-coat fusion proteins. This manner of expression isadvantageous for affinity-sharpening of antibodies.

Humanized Antibodies

Humanized antibodies may also be used in combination with the HSAlinker, in which one or more of the antibody CDRs are derived from anon-human antibody sequence, and one or more, but preferably all, of theCDRs bind specifically to an antigen (e.g., a protein, glycoprotein, orother suitable epitope).

A humanized antibody contains constant framework regions derivedsubstantially from a human antibody (termed an acceptor antibody), aswell as, in some instances, a majority of the variable region derivedfrom a human antibody. One or more of the CDRs (all or a portionthereof, as well as discreet amino acids surrounding one or more of theCDRs) are provided from a non-human antibody, such as a mouse antibody.The constant region(s) of the antibody, may or may not be present.

The substitution of one or more mouse CDRs into a human variable domainframework is most likely to result in retention of their correct spatialorientation if the human variable domain framework adopts the same or asimilar conformation as the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence and structural identity with the murine variable frameworkdomains from which the CDRs were derived. The heavy and light chainvariable framework regions can be derived from the same or differenthuman antibody sequences. The human antibody sequences can be thesequences of naturally occurring human antibodies, consensus sequencesof several human antibodies, or can be human germline variable domainsequences. See, e.g., Kettleborough et al., Protein Engineering 4:773(1991); Kolbinger et al., Protein Engineering 6:971 (1993).

Suitable human antibody sequences are identified by alignments of theamino acid sequences of the mouse variable regions with the sequences ofknown human antibodies. The comparison is performed separately for heavyand light chains but the principles are similar for each.

Methods of preparing chimeric and humanized antibodies and antibodyfragments are described in, e.g., U.S. Pat. Nos. 4,816,567, 5,530,101,5,622,701, 5,800,815, 5,874,540, 5,914,110, 5,928,904, 6,210,670,6,677,436, and 7,067,313 and U.S. Patent Application Nos. 2002/0031508,2004/0265311, and 2005/0226876. Preparation of antibody or fragmentsthereof is further described in, e.g., U.S. Pat. Nos. 6,331,415,6,818,216, and 7,067,313.

Receptors and Ligands

Within certain HSA linker conjugates, protein or glycoprotein receptorsor ligands are bound to an HSA linker. HSA linkers bonded with areceptor or ligand can be used, for example, to specifically target asecreted protein, a cell (e.g., a cancer cell), tissue, or organ.Furthermore, the specific binding of the HSA linker-receptor or -ligandconjugate to cognate target receptors or ligands can cause agonistic orantagonistic biological activity in intracellular or intercellularsignaling pathways. As with the other binding moieties described herein,receptors and ligands, or fragments thereof, can be conjoined to theamino and/or carboxy termini of an HSA linker, to a peptide connectorlinked to the HSA linker or to an amino acid residue within the HSAlinker.

Exemplary receptors and ligands that can be joined to an HSA linkerinclude, but are not limited to, insulin-like growth factor 1 receptor(IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelialtransition factor receptor (c-met; also known as hepatocyte growthfactor receptor (HGFR)), hepatocyte growth factor (HGF), epidermalgrowth factor receptor (EGFR), epidermal growth factor (EGF), heregulin,fibroblast growth factor receptor (FGFR), platelet-derived growth factorreceptor (PDGFR), platelet-derived growth factor (PDGF), vascularendothelial growth factor receptor (VEGFR), vascular endothelial growthfactor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosisfactor alpha (TNF-α), TNF-β, folate receptor (FOLR), folate, transferrinreceptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, α4integrin, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR),hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD4, CD11,CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor),CD106 (vascular cell adhesion molecule 1 (VCAM1), CD166 (activatedleukocyte cell adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253(TNF-related apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2,CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin1 (IL-1), CTLA-4, receptors alpha and beta, MART-1, gp100, MAGE-1,ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1(MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^(Y), MUC-1, epithelialcell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostatespecific membrane antigen (PSMA), TAG-72 antigen, andbiologically-active fragments thereof.

Receptors and ligands can be expressed, isolated, or joined to an HSAlinker using any of the methods described supra.

Diagnostic Agents

The HSA linker, or any binding moiety conjugated thereto (e.g.,antibody, antibody fragment, receptor, or ligand), can be coupled to achelating agent or to a detectable label to form a diagnostic agent.Also contemplated are HSA linker conjugates that include a detectablelabel, as described herein, as well as one or more of the therapeuticagents or binding moieties described herein.

The HSA linker (or HSA linker conjugate) and chelator components can becoupled by reacting the free amino group of a threonine residue of theHSA linker (or HSA linker conjugate) with an appropriate functionalgroup of the chelator, such as a carboxyl group or activated ester. Forexample, a such coupling may be achieved by incorporating the chelatorethylenediaminetetraacetic acid (EDTA), which is common in the art ofcoordination chemistry, when functionalized with a carboxyl substituenton the ethylene chain. Synthesis of EDTA derivatives of this type arereported in Arya et al. (Bioconjugate Chemistry 2:323 (1991)), whichdescribes blocking each of the four coordinating carboxyl groups with at-butyl group while the carboxyl substituent on the ethylene chain isfree to react with the amino group of a peptide portion of the agent.

An HSA linker or an HSA linker conjugate may incorporate a metalchelator component that is peptidic, i.e., compatible with solid-phasepeptide synthesis. In this case, the chelator may be coupled in the samemanner as EDTA described above or, more conveniently, the chelator andHSA linker or HSA linker conjugate are synthesized in toto starting fromthe C-terminal residue of the HSA linker or HSA linker conjugate andending with the N-terminal residue of the chelator.

An HSA linker or an HSA linker conjugate may further incorporate alinking group component that serves to couple the HSA linker to thechelator while not adversely affecting the biological properties of theHSA linker, the targeting function of the binding moiety portion(s) ofthe HSA linker conjugate, or the metal binding function of the chelator.Suitable linking groups include amino acid chains and alkyl chainsfunctionalized with reactive groups for coupling to the HSA linker orthe HSA linker conjugate and to the chelator. An amino acid chain is thepreferred linking group when the chelator is peptidic so that the HSAlinker or HSA linker conjugate can be synthesized in toto by solid-phasetechniques. An alkyl chain-linking group may be incorporated in the HSAlinker or HSA linker conjugate by reacting the amino group of athreonine residue of a peptide portion of an HSA linker with a firstfunctional group on the alkyl chain, such as a carboxyl group or anactivated ester. Subsequently the chelator is attached to the alkylchain to complete the formation of the HSA linker or HSA linkerconjugate by reacting a second functional group on the alkyl chain withan appropriate group on the chelator. The second functional group on thealkyl chain is selected from substituents that are reactive with afunctional group on the chelator while not being reactive with athreonine residue of the mutated HSA linker. For example, when thechelator incorporates a functional group such as a carboxyl group or anactivated ester, the second functional group of the alkyl chain-linkinggroup can be an amino group. It will be appreciated that formation ofthe HSA linker or HSA linker conjugate may require protection anddeprotection of the functional groups present in order to avoidformation of undesired products. Protection and deprotection areaccomplished using protecting groups, reagents, and protocols common inthe art of organic synthesis. Particularly, protection and deprotectiontechniques employed in solid phase peptide synthesis described above maybe used.

An alternative chemical linking group to an alkyl chain is polyethyleneglycol (PEG), which is functionalized in the same manner as the alkylchain described above for incorporation in the HSA linker or HSA linkerconjugate. It will be appreciated that linking groups may alternativelybe coupled first to the chelator and then to the HSA linker or HSAlinker conjugate.

In one aspect, an HSA linker or HSA linker conjugate is coupled to adiagnostically useful metal capable of forming a complex. Suitablemetals include, e.g., radionuclides, such as technetium and rhenium intheir various forms (e.g., ^(99 m)TcO³⁺, ^(99 m)TcO₂ ⁺, ReO³⁺, and ReO₂⁺). Incorporation of the metal within the HSA linker or HSA linkerconjugate can be achieved by various methods common in the art ofcoordination chemistry. When the metal is technetium-99 m, the followinggeneral procedure may be used to form a technetium complex. An HSAlinker-chelator conjugate solution is formed initially by dissolving theHSA linker or HSA linker conjugate in aqueous alcohol such as ethanol.The solution is then degassed to remove oxygen then thiol protectinggroups are removed with a suitable reagent, for example, with sodiumhydroxide, and then neutralized with an organic acid, such as aceticacid (pH 6.0-6.5). In the labeling step, a stoichiometric excess ofsodium pertechnetate, obtained from a molybdenum generator, is added toa solution of the conjugate with an amount of a reducing agent such asstannous chloride sufficient to reduce technetium and heated. Thelabeled HSA linker or HSA linker conjugate may be separated fromcontaminants ^(99 m)TcO₄ ⁻ and colloidal ^(99 m)TcO₂chromatographically, for example, with a C-18 Sep Pak cartridge.

In an alternative method, labeling of an HSA linker can be accomplishedby a transchelation reaction. The technetium source is a solution oftechnetium complexed with labile ligands facilitating ligand exchangewith the selected chelator. Suitable ligands for transchelation includetartarate, citrate, and heptagluconate. In this instance the preferredreducing reagent is sodium dithionite. It will be appreciated that theHSA linker or HSA linker conjugate may be labeled using the techniquesdescribed above, or alternatively the chelator itself may be labeled andsubsequently coupled to an HSA linker to form an HSA linker-chelatorconjugate; a process referred to as the “prelabeled ligand” method.

Another approach for labeling an HSA linker, or any agent conjugatedthereto, involves immobilizing the HSA linker-chelator conjugate on asolid-phase support through a linkage that is cleaved upon metalchelation. This is achieved when the chelator is coupled to a functionalgroup of the support by one of the complexing atoms. Preferably, acomplexing sulfur atom is coupled to the support which is functionalizedwith a sulfur protecting group such as maleimide.

When labeled with a diagnostically useful metal, an agent that includesan HSA linker-chelator conjugate can be used to detect tissue at risk ofdeveloping cancer (e.g., lung cancer, breast cancer, colon cancer, andprostate cancer), age-related diseases (e.g., cardiovascular disease,cerebrovascular disease, or Alzheimer's disease), tobacco-relateddiseases (e.g., emphysema, aortic aneurysms, esophageal cancer, orsquamous cell cancer of the head and neck) by procedures established inthe art of diagnostic imaging. An agent that incorporates an HSA linkerlabeled with a radionuclide metal, such as technetium-99 m, may beadministered to a mammal (e.g., a human) by intravenous injection in apharmaceutically acceptable solution, such as isotonic saline, or byother methods described herein. The amount of a labeled agentappropriate for administration is dependent upon the distributionprofile of the chosen HSA linker or HSA linker conjugate in the sensethat an agent that incorporates a rapidly cleared HSA linker or HSAlinker conjugate may be administered at higher doses than an agent thatincorporates an HSA linker or HSA linker conjugate that clears lessrapidly. Unit doses acceptable for imaging tissues are in the range ofabout 5-40 mCi for a 70 kg individual. The in vivo distribution andlocalization of an agent that incorporates a labeled HSA linker or HSAlinker conjugate can be tracked by standard techniques described hereinat an appropriate time subsequent to administration, typically between30 minutes and 180 minutes and up to about 5 days depending upon therate of accumulation at the target site with respect to the rate ofclearance at non-target tissue.

An HSA linker, or any molecule or moiety conjugated thereto, can also bemodified or labeled to facilitate diagnostic or therapeutic uses.Detectable labels such as a radioactive, fluorescent, heavy metal, orother molecules may be bound to any of the agents. Single, dual, ormultiple labeling of an agent may be advantageous. For example, duallabeling with radioactive iodination of one or more residues combinedwith the additional coupling of, for example, ⁹⁰Y via a chelating groupto amine-containing side or reactive groups, would allow combinationlabeling. This may be useful for specialized diagnostic needs such asidentification of widely dispersed small neoplastic cell masses.

An HSA linker, or any molecule or moiety conjugated thereto, can also bemodified, for example, by halogenation of the tyrosine residues of thepeptide component. Halogens include fluorine, chlorine, bromine, iodine,and astatine. Such halogenated agents may be detectably labeled, e.g.,if the halogen is a radioisotope, such as, for example, ¹⁸F, ⁷⁵Br, ⁷⁷Br,¹²² I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At. Halogenated agentscontain a halogen covalently bound to at least one amino acid, andpreferably to D-Tyr residues in each agent molecule. Other suitabledetectable modifications include binding of other compounds (e.g., afluorochrome such as fluorescein) to a lysine residue of the agent, oranalog, particularly an agent or analog having a linker includinglysines.

Radioisotopes for radiolabeling an HSA linker, or any molecule or moietyconjugated thereto, include any radioisotope that can be covalentlybound to a residue of the peptide component of the agent or analogthereof. The radioisotopes can also be selected from radioisotopes thatemit either beta or gamma radiation, or alternatively, any of the agentscan be modified to contain chelating groups that, for example, can becovalently bonded to lysine residue(s) of the HSA linker or any peptidicagent conjugated thereto. The chelating groups can then be modified tocontain any of a variety of radioisotopes, such as gallium, indium,technetium, ytterbium, rhenium, or thallium (e.g., ¹²⁵I, ⁶⁷Ga, ¹¹¹In,⁹⁹mTc, ¹⁶⁹Yb, ¹⁸⁶Re).

An HSA linker, or any molecule or moiety conjugated thereto, can bemodified by attachment of a radioisotope. Preferable radioisotopes arethose having a radioactive half-life corresponding to, or longer than,the biological half-life of the HSA conjugate used. More preferably, theradioisotope is a radioisotope of a halogen atom (e.g. a radioisotope offluorine, chlorine, bromine, iodine, and astatine), even more preferably⁷⁵Br, ⁷⁷Br, ⁷⁶Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At.

An agent that incorporates an HSA linker, or any molecule or moietyconjugated thereto, can be coupled to radioactive metals and used inradiographic imaging or radiotherapy. Preferred radioisotopes alsoinclude ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In ¹⁶⁸Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm,¹⁵⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²³Biand ²¹⁴Bi. The choice of metal is determined based on the desiredtherapeutic or diagnostic application.

An HSA linker, or any molecule or moiety conjugated thereto, can becoupled to a metal component, to produce a diagnostic or therapeuticagent. A detectable label may be a metal ion from heavy elements or rareearth ions, such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺. Agents that incorporatean HSA linker having paramagnetic or superparamagnetic metals conjoinedthereto are useful as diagnostic agents in MRI imaging applications.Paramagnetic metals include, but are not limited to, chromium (III),manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper(II), praseodymium (III), neodymium (III), samarium (III), gadolinium(III), terbium (III), dysprosium (III), holmium (III), erbium (III), andytterbium (III).

Chelating groups may be used to indirectly couple detectable labels orother molecules to an HSA linker or to an agent conjugated thereto.Chelating groups can link agents with radiolabels, such as abifunctional stable chelator, or can be linked to one or more terminalor internal amino acid reactive groups. An HSA linker, or any moleculeor moeity conjugated thereto, can be linked via an isothiocyanate β-Alaor appropriate non-α-amino acid linker which prevents Edman degradation.Examples of chelators known in the art include, for example, theininocarboxylic and polyaminopolycarboxylic reactive groups,ininocarboxylic and polyaminopolycarboxylic reactive groups,diethylenetriaminepentaacetic acid (DTPA), and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

An HSA linker, when expressed recombinantly, can be joined to a peptidicdetectable label or diagnostic agent. Peptides and proteins that can beused as a detectable label with an HSA linker include, but are notlimited to, fluorescent proteins, bioluminescent proteins, and epitopetags, each of which is discussed in detail below. One or more of thesedetectable labels can also be incorporated into an HSA linker conjugatethat also includes a therapeutic, cytotoxic, or cytostatic agent.

Fluorescent proteins or fluorochromes, such as green fluorescent protein(GFP; SEQ ID NO:47), enhanced GFP (eGFP), yellow fluorescent protein(SEQ ID NO:48; YFP), cyan fluorescent protein (SEQ ID NO:49; CFP), andred fluorescent protein (SEQ ID NO:50; RFP or DsRed), can be used asdetectable label joined to an HSA linker. Fluorescent proteins can berecombinantly expressed in a cell (e.g., a blood cell, such as alymphocyte) following transfection or transduction of the cell with anexpression vector that encodes the nucleotide sequence of thefluorescent protein. Upon exposure of the fluorescent protein to astimulating frequency of light, the fluorescent protein will emit lightat a low, medium, or high intensity that can be observed by eye under amicroscope or by an optical imaging device. Exemplary fluorescentproteins suitable for use as the diagnostic sequence in agents aredescribed in, e.g., U.S. Pat. Nos. 7,417,131 and 7,413,874, each ofwhich is herein incorporated by reference.

Bioluminescent proteins can also be used as a detectable labelincorporated into an HSA linker. Bioluminescent proteins, such asluciferase (e.g., firefly (SEQ ID NO:51), Renilla (SEQ ID NO:52), andOmphalotus luciferase) and aequorin, emit light as part of a chemicalreaction with a substrate (e.g., luciferin and coelenterazine). In oneembodiment, a vector encoding a luciferase gene provides for the invivo, in vitro, or ex vivo detection of cells (e.g., blood cells, suchas lymphocytes) that have been transduced or transfected according tostandard methods, such as those described herein. Exemplarybioluminescent proteins suitable for use as a diagnostic sequence andmethods for their use are described in, e.g., U.S. Pat. Nos. 5,292,658,5,670,356, 6,171,809, and 7,183,092, each of which is hereinincorporated by reference.

Epitope tags are short amino acid sequences, e.g., 5-20 amino acidresidues in length, that can be incorporated into an HSA linkerconjugate as a detectable label to facilitate detection once expressedin a cell, secreted from the cell, or bound to a target cell. An agentthat incorporates an epitope tag as a diagnostic sequence can bedetected by virtue of its interaction with an antibody, antibodyfragment, or other binding molecule specific for the epitope tag.Nucleotide sequences encoding the epitope tag are produced either bycloning appropriate portions of natural genes or by synthesizing apolynucleotide that encodes the epitope tag. An antibody, antibodyfragment, or other binding molecule that binds an epitope tag candirectly incorporate a detectable label (e.g., a fluorochrome,radiolabel, heavy metal, or enzyme such as horseradish peroxidase) orserve itself as a target for a secondary antibody, antibody fragment, orother binding molecule that incorporates such a label. Exemplary epitopetags that can be used as a diagnostic sequence include c-myc (SEQ IDNO:33), hemagglutinin (HA; SEQ ID NO:34), and histidine tag (His₆; SEQID NO:35). Furthermore, fluorescent (e.g., GFP) and bioluminescentproteins can also serve as epitope tags, as antibodies, antibodyfragments, and other binding molecules are commercially available forthe detection of these proteins.

The in vivo, in vitro, or ex vivo detection, imaging, or tracking of anHSA linker conjugate that incorporates a diagnostic sequence (e.g., afluorescent protein, bioluminescent protein, or epitope tag) or any cellexpressing or bound thereto can be accomplished using a microscope, flowcytometer, luminometer, or other state of the art optical imagingdevice, such as an IVIS® Imaging System (Caliper LifeSciences,Hopkinton, Mass.).

Therapeutic or Cytotoxic Agents Coupled to the HSA Linker

An HSA linker, or any molecule or moiety conjugated thereto, can becoupled to any known cytotoxic or therapeutic moiety to form an agent(an HSA linker conjugate) that can be administered to treat, inhibit,reduce, or ameliorate disease (e.g., a cancer, autoimmune disease, orcardiovascular disease) or one or more symptoms of disease. Examplesinclude but are not limited to antineoplastic agents such as: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin;aldesleukin; altretamine; ambomycin; a. metantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; camptothecin; caracemide; carbetimer;carboplatin; carmustine; carubicin hydrochloride; carzelesin;cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine;combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; daca (n-[2-(dimethyl-amino)ethyl]acridine-4-carboxamide);dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;dolasatins; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate;etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone;fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198;homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl;interferon alfa-n3; interferon beta-i a; interferon gamma-i b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; strontium chloride sr 89; sulofenur;talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine;tomudex; top53; topotecan hydrochloride; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2′deoxyformycin; 9-aminocamptothecin; raltitrexed;N-propargyl-5,8-dideazafolic acid;2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine;anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfurmustard; nitrogen mustard (mechlor ethamine); cyclophosphamide;melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea(MNU); N,N′-Bis (2-chloroethyl)-N-nitrosourea (BCNU);N-(2-chloroethyl)-N′ cyclohexyl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nitrosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin;carboplatin; ormaplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-aminocamptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

Other therapeutic compounds include, but are not limited to, 20-pi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycinB2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol;calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin);canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2′deoxycoformycin(DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epothilones (A, R=H; B,R=Me); epithilones; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide; etoposide4′-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide;homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin;idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mithracin;mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxinfibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; monoclonal antibody, human chorionic gonadotrophin;monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multipledrug resistance gene inhibitor; multiple tumor suppressor 1-basedtherapy; mustard anticancer agent; mycaperoxide B; mycobacterial cellwall extract; myriaporone; N-acetyldinaline; N-substituted benzamides;nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin;nartograstim; nedaplatin; nemorubicin; neridronic acid; neutralendopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxideantioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oralcytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin;paclitaxel analogues; paclitaxel derivatives; palauamine;palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin;pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium;pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol;phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetinB; plasminogen activator inhibitor; platinum complex; platinumcompounds; platinum-triamine complex; podophyllotoxin; porfimer sodium;porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan;topsentin; toremifene; totipotent stem cell factor; translationinhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growthinhibitory factor; urokinase receptor antagonists; vapreotide; variolinB; vector system, erythrocyte gene therapy; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

HSA linker conjugates can also include site-specifically conjugatedmolecules and moieties. Site-specific conjugation allows for thecontrolled stoichiometric attachment to specific residues in the HSAlinker of cytotoxic, immunomodulatory, or cytostatic agents including,e.g., anti tubulin agents, DNA minor groove binders, DNA replicationinhibitors, alkylating agents, anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy or radiation sensitizer, duocarmycins,etoposides, fluorinated pyrimidines, ionophores, lexitropsins,nitrosoureas, platinols, purine antimetabolites, puromycins, steroids,taxanes, topoisomerase inhibitors, and vinca alkaloids or any othermolecules or moieties described herein.

Techniques for conjugating therapeutic agents to proteins, and inparticular to antibodies, are well-known (e.g., Amon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy,” inMonoclonal Antibodies And Cancer Therapy (Reisfeld et al., eds., Alan R.Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (Robinson et al., eds., Marcel Dekker, Inc.,2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications (Pinchera et al., eds., 1985); “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled AntibodyIn Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy (Baldwin et al., eds., Academic Press, 1985); Thorpe et al.,Immunol. Rev. 62:119-58 (1982); and Doronina et al., “Development ofpotent monoclonal antibody auristatin conjugates for cancer therapy,”Nature Biotech. 21:(7)778-784 (2003)). See also, e.g., PCT publicationWO 89/12624.

An HSA linker, or any molecule or moiety conjugated thereto, can also becoupled to a lytic peptide. Such lytic peptides induce cell death andinclude, but are not limited to, streptolysin O; stoichactis toxin;phallolysin; staphylococcus alpha toxin; holothurin A; digitonin;melittin; lysolecithin; cardiotoxin; and cerebratulus A toxin (Kem etal., J. Biol. Chem. 253(16):5752-5757, 1978). An HSA linker, or anymolecule or moiety conjugated thereto (e.g., antibody or antibodyfragment conjugates), may be coupled to a synthetic peptide that sharessome sequence homology or chemical characteristics with any of thenaturally occurring peptide lysins; such characteristics include, butare not limited to, linearity, positive charge, amphipathicity, andformation of alpha-helical structures in a hydrophobic environment(Leuschner et al., Biology of Reproduction 73:860-865, 2005). An HSAlinker, or any molecule or moiety conjugated thereto, can also becoupled to an agent that induces complement-mediated cell lysis such as,for example, the immunoglobulin F_(c) subunit. An HSA linker, or anymolecule or moiety conjugated thereto, can also be coupled to any memberof the phospholipase family of enzymes (including phospholipase A,phospholipase B, phospholipase C, or phospholipase D) or to acatalytically-active subunit thereof.

An HSA linker, or any molecule or moiety conjugated thereto, can also becoupled to a radioactive agent to form an agent that can be used fordetection or therapeutic applications. Radioactive agents that can beused include but are not limited to Fibrinogen ¹²⁵I; Fludeoxyglucose¹⁸F; Fluorodopa ¹⁸F; Insulin ¹²⁵I; Insulin ¹³¹I; lobenguane ¹²³I;Iodipamide Sodium ¹³¹I; Iodoantipyrine ¹³¹I; Iodocholesterol ¹³¹I;lodohippurate Sodium ¹²³I; Iodohippurate Sodium ¹²⁵I; IodohippurateSodium ¹³¹I; Iodopyracet ¹²⁵I; Iodopyracet ¹³¹I; lofetamineHydrochloride ¹²³I; Iomethin ¹²⁵I; Iomethin ¹³¹I; ¹³¹I; IothalamateSodium ¹²⁵I; Iothalamate Sodium ¹³¹I tyrosine ¹³¹I; Liothyronine ¹²⁵I;Liothyronine ¹³¹I; Merisoprol Acetate ¹⁹⁷Hg; Merisoprol Acetate ²⁰³Hg;Merisoprol ¹⁹⁷Hg; Selenomethionine ⁷⁵Se; Technetium ^(99m)Tc AntimonyTrisulfide Colloid; Technetium ^(99m)Tc Bicisate; Technetium ^(99m)TcDisofenin; Technetium ^(99m)Tc Etidronate; Technetium ^(99m)TcExametazime; Technetium ^(99m)Tc Furifosmin; Technetium ^(99m)TcGluceptate; Technetium ^(99m)Tc Lidofenin; Technetium ^(99m)TcMebrofenin; Technetium ^(99m)Tc Medronate; Technetium ^(99m)Tc MedronateDisodium; Technetium ^(99m)Tc Mertiatide; Technetium ^(99m)TcOxidronate; Technetium ^(99m)Tc Pentetate; Technetium ^(99m)Tc PentetateCalcium Trisodium; Technetium ^(99m)Tc Sestamibi; Technetium ^(99m)TcSiboroxime; Technetium ^(99m)Tc; Succimer; Technetium ^(99m)Tc SulfurColloid; Technetium ^(99m)Tc Teboroxime; Technetium ^(99m)TcTetrofosmin; Technetium ^(99m)Tc Tiatide; Thyroxine ¹²⁵I; Thyroxine¹³¹I; Tolpovidone ¹³¹I; Triolein ¹²⁵I; or Triolein ¹³¹I.

Additionally, a radioisotope can be site-specifically conjoined to anHSA linker or HSA linker conjugate. The available reactive groups couldbe used to conjugate site-specific bifunctional chelating agents forlabeling of radioisotopes, including ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99 m)Tc,¹¹¹In, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁸⁹Zr,²¹¹At, ²¹²Bi, ²¹³Bi, or ²²⁵Ac.

Therapeutic or cytotoxic agents incorporated into or coupled with an HSAlinker or an HSA linker conjugate may further include, for example,anti-cancer Supplementary Potentiating Agents, including, but notlimited to: tricyclic anti-depressant drugs (e.g., imipramine,desipramine, amitryptyline, clomipramine, trimipramine, doxepin,nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclicanti-depressant drugs (e.g., sertraline, trazodone, and citalopram);Ca²⁺ antagonists (e.g., verapamil, nifedipine, nitrendipine, andcaroverine); Calmodulin inhibitors (e.g., prenylamine,trifluoroperazine, and clomipramine); Amphotericin B; Triparanol analogs(e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g.,buthionine and sulfoximine) and Multiple Drug Resistance reducing agentssuch as Cremaphor EL.

An agent that includes an HSA linker, or any molecule or moietyconjugated thereto, can also be coupled to or administered with one ormore cytokines (e.g., granulocyte colony stimulating factor,interferon-alpha, and tumor necrosis factor-alpha). An HSA linker, orany molecule or moiety conjugated thereto, can also be coupled to anantimetabolic agent. Antimetabolic agents include, but are not limitedto, the following compounds and their derivatives: azathioprine,cladribine, cytarabine, dacarbazine, fludarabine phosphate,fluorouracil, gencitabine chlorhydrate, mercaptopurine, methotrexate,mitobronitol, mitotane, proguanil chlorohydrate, pyrimethamine,raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate,vincristine sulfate, etc. More preferably, an HSA linker or conjugatecan be coupled to a folic acid-type antimetabolite, a class of agentsthat includes, for example, methotrexate, proguanil chlorhydrate,pyrimethanime, trimethoprime, or trimetrexate glucuronate, orderivatives of these compounds.

An HSA linker, or any molecule or moiety conjugated thereto, can also becoupled to a member of the anthracycline family of neoplastic agents,including but not limited to aclarubicine chlorhydrate, daunorubicinechlorhydrate, doxorubicine chlorhydrate, epirubicine chlorhydrate,idarubicine chlorhydrate, pirarubicine, or zorubicine chlorhydrate; acamptothecin, or its derivatives or related compounds, such as 10, 11methylenedioxycamptothecin; or a member of the maytansinoid family ofcompounds, which includes a variety of structurally-related compounds,e.g., ansamitocin P3, maytansine, 2′-N-demethylmaytanbutine, andmaytanbicyclinol.

An HSA linker, or any molecule or moiety conjugated thereto, can becoupled directly to a cytotoxic or therapeutic agent using knownchemical methods, or coupled indirectly to a cytotoxic or therapeuticagent via an indirect linkage. For example, an HSA linker can beattached to a chelating group that is attached to the cytotoxic ortherapeutic agent. Chelating groups include, but are not limited to,ininocarboxylic and polyaminopolycarboxylic reactive groups,diethylenetriaminepentaacetic acid (DTPA), and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Forgeneral methods, see, e.g., Liu et al., Bioconjugate Chem. 12(4):653,2001; Cheng et al., WO 89/12631; Kieffer et al., WO 93/12112; Albert etal., U.S. Pat. No. 5,753,627; and WO 91/01144 (each of which are herebyincorporated by reference).

An HSA linker conjugate that includes, e.g., an HSA linker, one or morebinding moieties (with or without intervening peptide connectors, asdefined herein), and a therapeutic or cytotoxic agent, can bespecifically targeted by the binding moiety (e.g., antibody, antibodyfragment, or receptor/ligand) to a cell or tissue, thereby allowingselective destruction of the target cell or tissue to which the bindingmoiety is directed. For example, an HSA linker conjugate can be used totarget and destroy cancer cells of the lung, breast, prostate, and colonin order to prevent, stabilize, inhibit the progression of, or treatcancers originating in these organs when the HSA linker conjugateincludes a binding moiety that specifically binds to the cancer cells inthese organs. Also, for example, an HSA linker conjugate can be used totarget and destroy cells of the vasculature, brain, liver, kidney,heart, lung, prostate, colon, nasopharynx, oropharynx, larynx, bronchus,and skin in order to prevent, stabilize, inhibit the progression of, ortreat age-related, tobacco-related, or autoimmune diseases or conditionsrelating to these organs by targeting, in the case of autoimmune diseasefor example, autoreactive T cells (e.g., by binding to and agonizingtumor necrosis factor receptor 2 (TNFR2) present on the autoreactive Tcells).

An HSA linker, when expressed recombinantly, can be joined to acytotoxic polypeptide. Cytotoxic polypeptides, when brought into contactwith a target cell (e.g., a cancer cell), exerts cytotoxic or cytostaticeffects on the cell. For example, a cytotoxic polypeptide, when joinedwith an HSA linker, can induce events in a target cell upon binding ofthe target cell that leads to cell death through, for example,apoptosis, necrosis, or senescence. Alternatively, a cytotoxicpolypeptide joined with an HSA linker can interfere or inhibit normalcellular biological activities, such as division, metabolism, andgrowth, or abnormal cellular biological activities, such as metastasis.

For example, an HSA linker joined to caspase 3 will bind a target cell(e.g., a cancer cell) and undergoe endocytosis. Once internalized by thetarget cell, the caspase portion of the HSA linker conjugate caninitiate the pro-apoptotic caspase cascade, ultimately resulting in theapoptosis of the target cell.

In a preferred embodiment, an HSA linker conjugate includes a cytotoxicpolypeptide capable of killing a cancer cell. In another embodiment, thecytotoxic polypeptide inhibits the growth or metastasis of a cancercell. The cytotoxic polypeptide joined with an HSA linker can also beused to kill or inhibit the growth of cells associated with, necessaryfor, or beneficial to cancer growth, such as endothelial cells that formblood vessels that perfuse solid tumors.

In an embodiment, an HSA linker conjugate can include two or morecytotoxic polypeptides so as to modulate (e.g., increase) thespecificity, intensity, or duration of the cytotoxic or cytostaticeffect on a target cell (e.g., a cancer cell).

In another embodiment, the HSA linker is joined to an activatable formof cytotoxic polypeptide (e.g., a biologically-inactive pro-agent thatis capable of activation upon cleavage by an enzyme or drug). In thisembodiment, texposure (e.g., in vivo) of the cytotoxic polypeptidepro-agent to an enzyme or drug capable of cleaving the cytotoxicpolypeptide, renders the cytotoxic polypeptide biologically-active(e.g., cytotoxic or cytostatic). An example of a biologically-inactivecytotoxic polypeptide that can be converted to a biologically-activeform for use with an HSA linker is a procaspase (e.g., procaspase 8 or3). For example, the procaspase 8 domain of an HSA linker can be cleavedby TRAIL or FasL upon internalization by a target cell (e.g., a cancercell). Once cleaved, the biologically active caspase 8 can promoteapoptosis of the target cell.

In one embodiment, the cytotoxic polypeptide joined to an HSA linker caninclude a full-length peptide, polypeptide, or protein, orbiologically-active fragment thereof (e.g., a “death domain”), known tohave cytotoxic or cytostatic properties. Peptides, polypeptides, orproteins with cytotoxic or cytostatic properties can be altered (e.g.,by making amino acid substitutions, mutations, truncations, oradditions) to facilitate incorporation of the cytotoxic sequence into anagent as described herein. Desirable alterations include, for example,changes to the amino acid sequence that facilitate protein expression,longevity, cell secretion, and target cell toxicity.

The present invention also provides a nucleic acid molecule encoding acytotoxic polypeptide as a fusion protein with an HSA linker, optionallyincluding binding moieities and peptide connectors. The nucleic acidmolecule can be incorporated into a vector (e.g., an expression vector),such that, upon expression of the HSA linker in a cell transfected ortransduced with the vector, the cytotoxic polypeptide, HSA linker, andbinding moieties, if present, are operably linked (e.g., fused,contiguously-joined, or tethered together). Examples of peptides,polypeptides, and proteins that can be used as a cytotoxic polypeptideof the present invention include, but are not limited to,apoptosis-inducing proteins such as cytochrome c (SEQ ID NO:39);caspases (e.g., caspase 3 (SEQ ID NO:36) and caspase 8 (SEQ ID NO:37));procaspases, granzymes (e.g., granzymes A and B (SEQ ID NO:38)); tumornecrosis factor (TNF) and TNF receptor family members, includingTNF-alpha (TNFα; SEQ ID NO:40)), TNF-beta, Fas (SEQ ID NO:41) and Fasligand; Fas-associated death domain-like IL-1β converting enzyme(FLICE); TRAIL/APO2L (SEQ ID NO:45) and TWEAK/APO3L (see, e.g., U.S.Patent Application Publication No. 2005/0187177, herein incorporated byreference); pro-apoptotic members of the Bcl-2 family, including Bax(SEQ ID NO:46), Bid, Bik, Bad (SEQ ID NO:42), Bak, and RICK (see, e.g.,U.S. Patent Application Publication No. 2004/0224389, herein incorporateby reference); vascular apoptosis inducing proteins 1 and 2 (VAP1 andVAP2; Masuda et al., Biochem. Biophys. Res. Commun. 278:197-204 (2000));pierisin (SEQ ID NO:44; Watanabe et al., Biochemistry 96:10608-10613(1999)); apoptosis-inducing protein (SEQ ID NO:43; AIP; Murawaka et al.,Nature 8:298-307 (2001)); IL-1α propiece polypeptide (see, e.g., U.S.Pat. No. 6,191,269, herein incorporated by reference); apoptin andapoptin-associated proteins such as AAP-1 (see, e.g., European PatentApplication Publication No. EP 1083224, herein incorporated byreference); anti-angiogenic factors such as endostatin and angiostatin;and other apoptosis-inducing proteins, including those described in thefollowing International and U.S. Patent Application Publications, eachherein incorporated by reference: U.S. 2003/0054994, U.S. 2003/0086919,U.S. 2007/0031423, WO 2004/078112, WO 2007/012430, and WO 2006/0125001(intracellular domain of delta 1 and jagged 1).

Wild-Type HSA Linker Conjugates

The present invention also encompasses a naturally-occurring wild-typeHSA linker, the amino acid and nucleotide sequences of which areprovided in SEQ ID NOS:3 and 4, respectively, in the formation ofbinding, diagnostic, or therapeutic agents. In all embodiments utilizingan HSA linker with the amino acid sequence listed in SEQ ID NO:3, one ormore peptide connectors, as described above, are covalently attached tothe amino and/or carboxy termini of the HSA linker, or to an amino acidresidue within the HSA linker sequence, to facilitate conjugation of oneor more binding moieties.

Truncations

The invention further provides an HSA linker conjugate that is formedusing a truncated wild-type HSA polypeptide, optionally combined withone or more peptide connectors or binding moieties. A wild-type HSApolypeptide lacking 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 200 or moreamino acids of the full-length wild-type HSA amino acid sequence (i.e.,SEQ ID NO:3) can be conjoined to any of the binding moieties ordiagnostic or therapeutic agents described herein. Truncations can occurat one or both ends of the HSA linker, or can include a deletion ofinternal residues. Truncation of more than one amino acid residue neednot be linear (i.e., consecutive). Examples of wild-type HSA linkersinclude those having, in combination with one or more peptide connectorsor binding moieties, one or more of domain I (SEQ ID NO:56; residues1-197 of SEQ ID NO:3), domain II (SEQ ID NO:54; residues 189-385 of SEQID NO:3), or domain III (SEQ ID NO:57; residues 381-585 of SEQ ID NO:3),or combinations thereof, e.g., domains I and II, I and III, and II andIII.

Serum clearance rates of a conjugate (e.g., a bispecific HSA-drug orradioisotope-containing agent), can be optimized by testing conjugatescontaining a truncated wild-type HSA linker, as described above.

Additional HSA Linker Modifications

HSA linkers may, but need not, be modified by site-specific chemicalmodification of amino acid residues in the HSA linker. Thecorrectly-folded tertiary structure of HSA displays certain amino acidresidues on the external face of the protein. Chemically-reactive aminoacid residues (e.g., cysteine) can be substituted for thesesurface-exposed residues to allow site-specific conjugation of adiagnostic or therapeutic agent.

Alternatively, or in addition, HSA linkers may optionally be modified bythe addition or removal of asparagine, serine, or threonine residuesfrom an HSA linker sequence to alter glycosylation of these amino acidresidues. Glycosylation sites added to an HSA linker are preferablysurface-exposed, as discussed herein. Glycosyl or other carbohydratemoieties introduced to an HSA linker can be directly conjugated todiagnostic, therapeutic, or cytotoic agents.

Cysteine (Thiol) Conjugation

Surface-exposed amino acid residues of the HSA linker may be substitutedwith cysteine residues to allow for chemical conjugation of diagnostic,therapeutic, or cytotoxic agents. Cysteine residues exposed on thesurface of the HSA linker (when folded into its native tertiarystructure) allow the specific conjugation of a diagnostic, therapeutic,or cytotoxic agent to a thiol reactive group such as maleimide orhaloacetyl. The nucleophilic reactivity of the thiol functionality of acysteine residue to a maleimide group is about 1000 times highercompared to any other amino acid functionality in a protein, such as theamino group of a lysine residue or the N-terminal amino group. Thiolspecific functionality in iodoacetyl and maleimide reagents may reactwith amine groups, but higher pH (>9.0) and longer reaction times arerequired (Garman, 1997, Non-Radioactive Labelling: A Practical Approach,Academic Press, London). The amount of free thiol in a protein may beestimated using the standard Ellman's assay. In some instances,reduction of the disulfide bonds with a reagent such as dithiothreitol(DTT) or selenol (Singh et al., Anal. Biochem. 304:147-156 (2002)) isrequired to generate the reactive free thiol.

Sites for cysteine substitution can be identified by analysis of surfaceaccessibility of the HSA linker (e.g., the identification of serine andthreonine residues as suitable for substitution are described in Example1 below). The surface accessibility can be expressed as the surface area(e.g., square angstroms) that can be contacted by a solvent molecule,e.g., water. The occupied space of water is approximated as a spherewith a 1.4 angstrom radius. Software for calculating the surfaceaccessibility of each amino acid of a protein is freely available orlicensable. For example, the CCP4 Suite of crystallography programswhich employ algorithms to calculate the surface accessibility of eachamino acid of a protein with known x-ray crystallography derivedcoordinates (“The CCP4 Suite: Programs for Protein Crystallography”Acta. Cryst. D50:760-763 (1994); www.ccp4.ac.uk/dist/html/INDEX.html).Solvent accessibility may also be assessed using the free softwareDeepView Swiss PDB Viewer downloaded from the Swiss Institute ofBioinformatics. The substitution of cysteines at surface-exposed sitesallows for conjugation of the reactive cysteine to a thiol reactivegroup linked to the diagnostic or therapeutic agent.

Glycosylation

In addition, altered serum clearance rates can be achieved byengineering glycosylation sites into the HSA linker. In certainembodiments, an HSA linker is glycosylated. Glycosylation ofpolypeptides is typically either N-linked or O-linked. N-linked refersto the attachment of a carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X represents any amino acid exceptproline, are the recognition sequences for enzymatic attachment of thecarbohydrate moiety to the asparagine side chain. Thus, the presence ofeither of these tripeptide sequences in a polypeptide creates apotential glycosylation site. O-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the HSA linker isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created. The alteration may also be made by theaddition, deletion, or substitution of one or more serine or threonineresidues to the sequence of the original HSA linker (for O-linkedglycosylation sites). The resulting carbohydrate structures on HSA canalso be used for site-specific conjugation of cytotoxic,immunomodulatory or cytostatic agents as described above.

HSA Linker Conjugates in Combination with Other Therapeutic Agents

HSA linker conjugates described herein may be administered with one ormore of the therapeutic, cytotoxic, or cytostatic agents describedherein. For example, a patient suffering from breast cancer can beadministered an HSA linker containing ErbB2 and ErbB3 scFvs (e.g.,B2B3-1) can be co-administered with, e.g., doxorubicin,cyclophosphamide, and paclitaxel, a common chemotherapeutic regimen forthe treatment of breast cancer. A preferred therapeutic agent for use inthis regard is trastuzumab. Data regarding this combination are setforth in Examples 42-44 below. Additional biological and chemical agentsuseful for the treatment of cancer are set forth herein, e.g., inAppendix 2.

HSA Linker Conjugates in Combination with Radiotherapy or Surgery

HSA linker conjugates may be administered prior to, concurrent with, orfollowing radiotherapy or surgery. For example, a patient suffering froma proliferative disorder (e.g., breast cancer) can receive an HSA linkerconjugate, alone or in combination with other therapeutic, cytotoxic, orcytotoxic agents as described herein concurrent with targetedradiotherapy or surgical intervention (e.g., lumpectomy or mastectomy)at the site of the cancerous tissue. Radiotherapies suitable for use incombination with HSA linker conjugates include brachytherapy andtargeted intraoperative radiotherapy (TARGIT).

Pharmaceutical Compositions

Pharmaceutical compositions provided herein contain a therapeutically ordiagnostically effective amount of an HSA linker conjugate that includesone or more of a binding moiety (e.g., antibodies or antibodyfragments), a diagnostic agent (e.g., radionuclide or chelating agents),or a therapeutic agent (e.g., cytotoxic or immunomodulatory agents)agent. The active ingredients, an HSA linker conjugate (prepared withone or more of a binding moiety, diagnostic agent, or therapeutic agent)can be formulated for use in a variety of drug delivery systems. One ormore physiologically acceptable excipients or carriers can also beincluded in the compositions for proper formulation. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). For a brief review of methods for drug delivery, see,Langer Science 249:1527-1533 (1990).

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. Commonly, thepharmaceutical compositions are administered parenterally (e.g., byintravenous, intramuscular, or subcutaneous injection), or by oralingestion, or by topical application. Thus, compositions for parenteraladministration may include an HSA linker, with or without one or morebinding, diagnostic, and/or therapeutic agent conjugated thereto,dissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a tablet, a capsule, andthe like. Furthermore, this invention provides compositions for localadministration, which may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of an HSA linker conjugate (prepared with oneor more of a binding, diagnostic, and/or therapeutic agent) in a sealedpackage of tablets or capsules, for example. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount of an HSA linkerconjugate (prepared with one or more of a binding, diagnostic, and/ortherapeutic agent) can be administered to a mammal (e.g., a human) forprophylactic and/or therapeutic treatments. In prophylacticapplications, compositions containing an HSA linker conjugate (preparedwith one or more of a binding, diagnostic, and/or therapeutic agent) areadministered to a patient susceptible to or otherwise at risk ofdeveloping a disease or condition (e.g., a cancer, autoimmune disease,or cardiovascular disease). Such an amount is defined to be a“prophylactically effective dose.” In this use, the precise amountsagain depend on the patient's state of health, but generally range fromabout 0.5 mg to about 400 mg of an HSA linker conjugate (prepared withone or more of a binding, diagnostic, and/or therapeutic agent) per dose(e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose)and from about 0.1 μg to about 300 mg of one or more immunomodulatoryagents per dose (e.g., 10 μg, 30 μg, 50 μg, 0.1 mg, 10 mg, 50 mg, 100mg, or 200 mg per dose). A dose of an HSA linker conjugate (preparedwith one or more of a binding, diagnostic, and/or therapeutic agent) canbe administered prophylactically to a patient one or more times perhour, day, week, month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or12 times per hour, day, week, month, or year). More commonly, a singledose per week of an HSA linker conjugate (prepared with one or more of abinding, diagnostic, and/or therapeutic agent) is administered.

In therapeutic applications, a dose of an HSA linker conjugate (preparedwith one or more of a binding, diagnostic, and/or therapeutic agent) isadministered to a mammal (e.g., a human) already suffering from adisease or condition (e.g., a cancer, autoimmune disease, orcardiovascular disease) in an amount sufficient to cure or at leastpartially arrest or alleviate one or more of the symptoms of the diseaseor condition and its complications. An amount adequate to accomplishthis purpose is defined as a “therapeutically effective dose.” Amountseffective for this use may depend on the severity of the disease orcondition and general state of the patient, but generally range fromabout 0.5 mg to about 400 mg of an HSA linker conjugate (prepared withone or more of a binding, diagnostic, and/or therapeutic agent) per dose(e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more perdose). A dose of an HSA linker conjugate (prepared with one or more of abinding, diagnostic, and/or therapeutic agent) can be administeredtherapeutically to a patient one or more times per hour, day, week,month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per hour,day, week, month, or year). More commonly, a single dose per week of anHSA linker conjugate (prepared with one or more of a binding,diagnostic, and/or therapeutic agent) is administered.

In several embodiments, the patient may receive an HSA linker conjugate(prepared with one or more of a binding, diagnostic, and/or therapeuticagent) in the range of about 0.5 to about 400 mg per dose one or moretimes per week (e.g., 2, 3, 4, 5, 6, or 7 or more per week), preferablyabout 5 mg to about 300 mg per dose one or more times per week, and evenmore preferably about 5 mg to about 200 mg per dose one or more timesper week. The patient may also receive a biweekly dose of an HSA linkerconjugate (prepared with one or more of a binding, diagnostic, and/ortherapeutic agent) in the range of about 50 mg to about 800 mg or amonthly dose of an HSA linker, or any binding, diagnostic, and/ortherapeutic agent conjugated thereto, in the range of about 50 mg toabout 1,200 mg.

In other embodiments, an HSA linker conjugate (prepared with one or moreof a binding, diagnostic, and/or therapeutic agent) may be administeredto a patient in a typical dosage range of about 0.5 mg per week to about2000 mg per week, about 1.0 mg per week to about 1000 mg per week, about5 mg per week to about 500 mg per week, about 10 mg per week to about100 mg per week, about 20 mg per week to about 80 mg per week, about 100mg per week to about 300 mg per week, or about 100 mg per week to about200 mg per week. In another aspect, the dosages for administration to a70 kg patient can range from, for example, about 1 μg to about 5000 mg,about 2 μg to about 4500 mg, about 3 μg to about 4000 mg, about 4 μg toabout 3,500 mg, about 5 μg to about 3000 mg, about 6 μg to about 2500mg, about 7 μg to about 2000 mg, about μg to about 1900 mg, about 9 μgto about 1,800 mg, about 10 μg to about 1,700 mg, about 15 μg to about1,600 mg, about 20 μg to about 1,575 mg, about 30 μg to about 1,550 mg,about 40 μg to about 1,500 mg, about 50 μg to about 1,475 mg, about 100μg to about 1,450 mg, about 200 μg to about 1,425 mg, about 300 μg toabout 1,000 mg, about 400 μg to about 975 mg, about 500 μg to about 650mg, about 0.5 mg to about 625 mg, about 1 mg to about 600 mg, about 1.25mg to about 575 mg, about 1.5 mg to about 550 mg, about 2.0 mg to about525 mg, about 2.5 mg to about 500 mg, about 3.0 mg to about 475 mg,about 3.5 mg to about 450 mg, about 4.0 mg to about 425 mg, about 4.5 mgto about 400 mg, about 5 mg to about 375 mg, about 10 mg to about 350mg, about 20 mg to about 325 mg, about 30 mg to about 300 mg, about 40mg to about 275 mg, about 50 mg to about 250 mg, about 100 mg to about225 mg, about 90 mg to about 200 mg, about 80 mg to about 175 mg, about70 mg to about 150 mg, or about 60 mg to about 125 mg, of an HSA linkerconjugate provided herein. Dosage regimen may be adjusted to provide theoptimum therapeutic response. In another aspect, an HSA linker conjugate(prepared with one or more of a binding, diagnostic, and/or therapeuticagent) may be administered in the range of about 0.5 mg every other dayto about 500 mg every other day, preferably about 5 mg every other dayto about 75 mg every other day, more preferably about 10 mg every otherday to about 50 mg every other day, and even more preferably 20 mg everyother day to about 40 mg every other day. An HSA linker conjugate(prepared with one or more of a binding, diagnostic, and/or therapeuticagent) may also be administered in the range of about 0.5 mg three timesper week to about 100 mg three times per week, preferably about 5 mgthree times per week to about 75 mg three times per week, morepreferably about 10 mg three times per week to about 50 mg three timesper week, and even more preferably about 20 mg three times per week toabout 40 mg three times per week.

In non-limiting embodiments of the methods of the present invention, anHSA linker conjugate (prepared with one or more of a binding,diagnostic, and/or therapeutic agent) is administered to a mammal (e.g.,a human) continuously for 1, 2, 3, or 4 hours; 1, 2, 3, or 4 times aday; every other day or every third, fourth, fifth, or sixth day; 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 times a week; biweekly; 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 times a month; bimonthly; 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 times every six months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 times a year; or biannually. An HSAlinker conjugate (prepared with one or more of a binding, diagnostic,and/or therapeutic agent) may be administered at different frequenciesduring a therapeutic regime. In additional embodiments, an HSA linkerconjugate (prepared with one or more of a binding, diagnostic, and/ortherapeutic agent) may be administered to a patient at the samefrequency or at a different frequency.

The amount of one or more diagnostic or therapeutic agents and an HSAlinker, or any agent conjugated thereto, required to achieve the desiredtherapeutic effect depends on a number of factors, such as the specificdiagnostic or therapeutic agent(s) chosen, the mode of administration,and clinical condition of the recipient. A skilled artisan will be ableto determine the appropriate dosages of one or more diagnostic ortherapeutic agents and an HSA linker, or any agent conjugated thereto,to achieve the desired results.

Single or multiple administrations of the compositions comprising aneffective amount of an HSA linker conjugate (prepared with one or moreof a binding, diagnostic, and/or therapeutic agent) can be carried outwith dose levels and pattern being selected by the treating physician.The dose and administration schedule can be determined and adjustedbased on the severity of the disease or condition in a mammal (e.g., ahuman), which may be monitored throughout the course of treatmentaccording to the methods commonly practiced by clinicians or thosedescribed herein.

An HSA linker conjugate (prepared with one or more of a binding,diagnostic, and/or therapeutic agent) can be administered to a mammaliansubject, such as a human, directly or in combination with anypharmaceutically acceptable carrier or salt known in the art.Pharmaceutically acceptable salts may include non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and described,for example, in Remington's Pharmaceutical Sciences, (18^(th) edition),ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

Diagnostic and Therapeutic Applications

HSA linker conjugates can be used for diagnostic and therapeuticapplications in a human, including, for example, the diagnosis ortreatment of proliferative diseases (e.g., cancers, such as melanoma,clear cell sarcoma, and renal cancer) and autoimmune diseases (e.g.,multiple sclerosis, rheumatoid arthritis, and uveitis). The followingdiscussion of human proliferative and autoimmune diseases is meant toprovide the skilled practitioner with a general understanding of how HSAlinker conjugates can be applied in diagnostic and therapeuticapplications and is not meant to limit the scope of the presentinvention.

Proliferative Diseases (Cancer)

An HSA linker conjugate can be used to diagnose, treat, prevent, oreliminate proliferative diseases such as, but not limited to, breastcancer, melanoma, clear cell sarcoma, renal cancer (e.g., renal cellcarcinoma), prostate cancer, lung cancer, gastric cancer, and ovariancancer. Binding moieties to be conjoined with an HSA linker fordiagnostic or therapeutic application in a patient suspected of havingor suffering from a proliferative disease may be chosen based on theirability to specifically bind, agonize, activate, antagonize, or inhibittarget molecules (e.g., cell surface receptors such as tyrosine kinasereceptors) associated with a proliferative disease. Binding moietiesthat target, for example, insulin-like growth factor receptor (IGFR,e.g., IGF1R and IGF2R), fibroblast growth factor receptor (FGFR),platelet-derived growth factor receptor (PDGFR), vascular endothelialgrowth factor receptor (VEGFR), tumor necrosis factor receptor (TNFR),epidermal growth factor receptor (EGFR, e.g., ErbB2 (HER2/neu)), Fcreceptor, c-kit receptor, or mesenchymal epithelial transition factorreceptor (c-met; also known as hepatocyte growth factor receptor (HGFR))can be conjoined to an HSA linker to diagnose or treat a proliferativedisease. Specific binding of a cancer cell by an HSA linker conjugatecan allow for detection (e.g., an HSA linker conjoined to a detectablelabel, as defined herein) or destruction (e.g., an HSA linker conjoinedto a cytotoxic agent) of the bound cancer cell. Specific application ofHSA linker conjugates for the treatment of breast and renal cancer isdescribed below.

Breast Cancer

Common forms of breast cancer include invasive ductal carcinoma, amalignant cancer in the breast's ducts, and invasive lobular carcinoma,a malignant cancer in the breast's lobules. Some types of breast cancercells are known to express high levels of epidermal growth factorreceptors, especially ErbB2 (i.e., HER2/neu). Aberrant signaling orunregulated activation of EGFRs has been linked to the development andprogression of many cancers, including breast cancer. Uncontrolledcellular proliferation mediated via dysfunctional EGFR pathways can befound in a wide variety of solid tumors of epithelial origin and datahave linked tumor EGFR expression, overexpression, and dysregulation toadvanced disease, metastatic phenotype, resistance to chemotherapy, andan overall poorer prognosis.

An HSA linker conjoined to one or more binding moieties specific for anEGFR (e.g., anti-ErbB2; trastuzumab) can be used with a diagnostic(e.g., a detectable label) or cytotoxic, cytostatic, or therapeuticagent, as described herein, to diagnose or treat breast cancer.Alternatively, a bispecific HSA linker conjugate that comprises bindingmoieties specific for ErbB2 and ErbB3, such as “B2B3-1,” describedfurther herein, can be employed to diagnose or treat cancers, e.g.,breast, kidney, ovarian, and lung cancers.

As described above, an HSA linker conjugate used to treat breast cancercan be administered prior to (e.g., neoadjuvant chemotherapy),concurrent with, or following (e.g., adjuvant chemotherapy) radiotherapyor surgical intervention. An HSA linker conjugate can also beco-administered with other compounds (e.g., antineoplastic agents, suchas biological or chemical therapeutics) useful for the treatment ofbreast cancer. For example, the antineoplastic agents listed in Table 1,including mitotic inhibitors (e.g., taxanes), topoisomerase inhibitors,alkylating agents (including, e.g., platinum-based agents), selectiveestrogen modulators (SERM), aromatase inhibitors, antimetabolites,antitumor antibiotics (e.g., anthracycline antibiotics), anti-VEGFagents, anti-ErbB2 (HER2/neu) agents, and anti-ErbB3 agents, are knownto be particularly useful for the treatment of breast cancer. An HSAlinker conjugate can be administered by a clinician in combination withany compound, including those listed in Appendix 2, known or thought tobe beneficial for the treatment of breast cancer.

TABLE 1 Exemplary antineoplastic agents for treatment of breast cancerin combination with HSA linker conjugates. Exemplary Agent TherapeuticClass (Generic/Tradename) Exemplary Dose Mitotic Inhibitors paclitaxel(TAXOL ®; 175 mg/m² ABRAXANE ®) docetaxel (TAXOTERE ®) 60-100 mg/m²Topoisomerase Inhibitors camptothecin topotecan hydrochloride(HYCAMTIN ®) etoposide (EPOSIN ®) Alkylating Agents cyclophosphamide(CYTOXAN ®) 600 mg/m² Platinum-Based Agents Cisplatin 20-100 mg/m²carboplatin (PARAPLATIN ®) 300 mg/m² nedaplatin (AQUPLA ®) oxaliplatin(ELOXATIN ®) 65-85 mg/m² satraplatin (SPERA ®) triplatin tetranitrateSelective Estrogen Modulators tamoxifen (NOLVADEX ®) 20-40 mg/day (SERM)raloxifene (EVISTA ®) 60 mg/day toremifene (FARESTON ®) Antimetabolitesmethotrexate 40 mg/m² Fluorouracil (5-FU) 500 mg/m² RaltitrexedAntitumor Antibiotics Doxorubicin (ADRIAMYCIN ®) 40-75 mg/m² epirubicin(ELLENCE ®) 60-120 mg/m² Aromatase Inhibitors aminoglutethimide(CYTADREN ® 250-2000 mg/day anastrozole (ARIMIDEX ®) 1 mg/day letrozole(FEMARA ®) 2.5 mg/day Vorozole exemestane (AROMASIN ®) 25-50 mg/dayTestolactone fadrozole (AFEMA ®) Anti-VEGF Agents bevacizumab(AVASTIN ®) 10 mg/kg Anti-ErbB2 (HER2/neu) Agents trastuzumab(HERCEPTIN ®) 2-8 mg/kg Pertuzumab (OMNITARG ®) Anti-ErbB3 (HER3) AgentsU3-1287 (AMG 888)

Renal Cancer

Kidney cancers, such as renal cell carcinoma, are particularly resistantto traditional radiological and chemical therapies. As such, theapplication of biological therapeutics, conjoined with an HSA linker,represents an attractive option for patients suffering from thesecancers. For example, an HSA linker conjoined with binding moieties thatagonize type I interferon or interleukin 2 receptors can be used totreat a renal cancer. As a solid tumor, binding moieties that target andinhibit tumor vascularization (e.g., anti-vascular endothelial growthfactor (VEGF) antibodies such as bevacizumab) can also be used fortherapeutic effect.

Autoimmune Diseases

An HSA linker conjugate can be used to diagnose, treat, prevent, orstabilize autoimmune diseases and disorders in e.g., a human patient,such as, e.g., multiple sclerosis (MS), insulin-dependent diabetesmellitus (IDDM), rheumatoid arthritis (RA), uveitis, Sjögren's syndrome,Grave's disease, psoriasis, and myasthenia gravis. Autoimmune diseasesand disorders are caused by self-reactive elements of the immune system(e.g., T cells, B cells, and self-reactive antibodies). As such, bindingmoieties that inhibit, block, antagonize, or deplete (e.g.,anti-lymphocyte or anti-thymocyte globulins; basiliximab, daclizumab, ormuromonab-CD3 monoclonal antibodies) self-reactive immune cells andantibodies can be conjoined with an HSA linker for therapeutic use.Binding moieties that function as inflammatory signaling inhibitors(ISI), as defined herein, can be conjoined to an HSA linker for thetreatment of autoimmunity. In addition, binding moieties that inhibit orantagonize integrin function (e.g., an integrin antagonist, as definedherein) can ameliorate or halt disease progression.

In other embodiments, the binding moiety is a soluble TNF receptor, suchas etanercept or lenercept; an antibody directed against apro-inflammatory cytokine or a pro-inflammatory cell surface signalingmolecule, such as adalimumab, certolizumab, inflixamab, golimumab, andrituxan; a dominant-negative pro-inflammatory cytokine variant, such asXENP345, XPRO™1595, anakinra, and variants disclosed in U.S. PatentApplication Publication Nos. 20030166559 and 20050265962; an inhibitorof the signaling pathways downstream of pro-inflammatory cytokine orpro-inflammatory cell surface signaling molecules, such as DE 096,5-amino-2-carbonylthiopene derivatives (as described in WO2004089929),ARRY-797, BIRB 796 BS,(1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(morpholin-4-yl-ethoxy)-naphtalen-1-yl]-urea,CHR-3620, CNI-1493, FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan),ISIS 101757 (Isis Pharmaceuticals), ML3404, NPC31145, PD169316, PHZ1112,RJW67657,4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl)-3-butyn-1-ol,SCIO-469, SB202190, SB203580,(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB239063,trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole,SB242235, SD-282, SKF-86002, TAK 715, VX702, and VX745; or an inhibitorof TNF-alpha converting enzyme (TACE), such as BB-1101, BB-3103,BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474,DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI005); or an anti-idiotypic agent, such as monoclonal antibodies, LJP 394(abetimus, RIQUENT®, La Jolla Pharmaceuticals).

In other embodiments, the binding moiety is an interferon, as describedherein. Binding moieties that can be conjoined to an HSA linker include,e.g., interferon-beta (REBIF® (IFN-β-1a), AVONEX® (IFN-β-1a), andBETASERON® (IFN-β-1b)), interferon-t (TAUFERON™), interferon-alpha(e.g., ROFERON-A® (IFN-α-2a), INTRON-A® (IFN-α-2b), REBETRON®(IFN-α-2b), ALFERON-N® (IFN-α-n3), PEG-INTRON® (IFN-α-2b covalentlyconjugated with monomethoxy polyethylene glycol), INFERGEN® (anon-naturally occurring type 1 interferon with 88% homology toIFN-α-2b), or PEGASYS® (pegylated IFN-α-1a)), and ACTIMMUNE® (IFN-g-1b).

The present invention further provides HSA linker conjugates withbinding moieties that antagonize these pro-inflammatory molecules ortheir specific receptors to treat autoimmunity. Specific application ofHSA linker conjugates for the diagnosis and treatment of MS and RA aredescribed below.

Multiple Sclerosis

Multiple sclerosis (MS) is a neurological disease characterized byirreversible degeneration of the nerves of the central nervous system(CNS). Although the underlying cause is unclear, the neurodegenerationin MS is the direct result of demyelination, or the stripping of myelin,a protein that normally lines the outer layer and insulates the nerves.T cells play a key role in the development of MS. Inflamed MS lesions,but not normal white matter, can have infiltrating CD4⁺ T cells thatrespond to self antigens presented by MHC class II-linked molecules suchas human HLA-DR2. The infiltrating CD4 T cells (T_(H)1 cells) producethe pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α that activateantigen-presenting cells (APCs) such as macrophages to produceadditional pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-8, andIL-12. IL-12 induces further IFN-γ synthesis. The result is progressivedemyelination of neuronal sheaths, leading to human disease.

HSA linker conjugates can be used to aid in the diagnosis of MS.Diagnostic HSA linker conjugates that include binding moieties thatspecifically target one or more (e.g., a bispecific HSA linkerconjugate) immune cell activation markers (e.g., CD69, CD28, HLA-DR, andCD45). An imbalance of one or more of these pro-inflammatory or immunecell activation mediators relative to other factors or cells may bemeasured using an HSA linker conjugate conjoined with a diagnostic agent(e.g., a radioisotope or fluorochrome).

An HSA linker conjugate can be used to treat a person at risk ofdeveloping or suffering from MS or to prevent, ameliorate, or cure thesymptoms of the disease. For example, binding moieties that specificallytarget and antagonize α4 integrin (e.g., natalizumab), CD52 (e.g.,alemtuzumab), CD80, P-selectin, sphingosine-1-phosphate receptor-1(S1PR1), hyaluronate receptor, leukocyte function antigen-1 (LFA-1),CD11 (e.g., efalizumab), CD18, CD20 (e.g., rituximab), CD85, ICOSligand, CCR2, CXCR3, or CCR5 can be useful when conjoined to an HSAlinker for therapeutic use in a patient suffering from MS. Similarly,binding moieties that neutralize type I interferons (e.g.,interferons-alpha and -beta) or that antagonize type I interferonreceptors (e.g., IFNαR1) can also be conjoined to an HSA linker fortherapeutic application.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorderthat causes the immune system to attack the joints. It is a disablingand painful inflammatory condition, which can lead to substantial lossof mobility due to pain and joint destruction. RA is a systemic disease,often affecting extra-articular tissues throughout the body includingthe skin, blood vessels, heart, lungs, and muscles.

Patients suffering from RA frequently have an increase in cellularexpression of the HLA-DR4/DR1 cluster. HSA linker conjugates specificfor one or both of these cell surface molecules are useful for thediagnosis of RA.

An HSA linker conjugate can be used to treat a person at risk ofdeveloping of suffering from RA to prevent, ameliorate, or cure thesymptoms of the disease. For example, binding moieties, as definedherein, that specifically target and antagonize TNF-α (e.g., etanercept,infliximab, and adalimumab), IL-1 (e.g., anakinra), or CTLA-4 (e.g.,abatacept). Binding moieties that specifically target and deplete Bcells (e.g., an anti-CD20 antibody, such as rituximab) can also beconjoined to the HSA linker described herein to treat or prevent RA.

Uveitis

Uveitis specifically refers to inflammation of the middle layer of theeye, but may refer to any inflammatory process involving the interior ofthe eye. Uveitis may be autoimmune or idiopathic in origin

An HSA linker conjugate can be used to treat a person at risk ofdeveloping of suffering from autoimmune uveitis to prevent, ameliorate,or cure the symptoms of the disease. For example, alpha-fetoprotein(e.g., human AFP; NCBI Accession No. NM_(—)001134), orbiologically-active fragments thereof, can be conjoined to an HSA linkerto reduce or eliminate inflammation associated with autoimmune oridiopathic uveitis.

Kits

The present invention further provides kits that include apharmaceutical composition containing an HSA linker, and one or more ofa binding moiety (e.g., antibodies or antibody fragments), a diagnosticagent (e.g., radionuclide or chelating agents), and a therapeutic agent(e.g., cytotoxic or immunomodulatory agents) with reagents that can beused to conjugate them to the HSA linker, if necessary, and including apharmaceutically-acceptable carrier, in a therapeutically effectiveamount for treating a disease or condition (e.g., a cancer, autoimmunedisease, or cardiovascular disease). The kits include instructions toallow a practitioner (e.g., a physician, nurse, or patient) toadminister the composition contained therein.

Preferably, the kits include multiple packages of the single-dosepharmaceutical composition(s) containing an effective amount of an HSAlinker, or any binding (e.g., antibodies or antibody fragments (e.g.,scFv)), diagnostic (e.g., radionuclide or chelating agents), and/ortherapeutic (e.g., cytotoxic or immunomodulatory agents) conjugatethereof. Optionally, instruments or devices necessary for administeringthe pharmaceutical composition(s) may be included in the kits. Forinstance, a kit may provide one or more pre-filled syringes containingan effective amount of an HSA linker, or any binding, diagnostic, and/ortherapeutic agent conjugated thereto. Furthermore, the kits may alsoinclude additional components such as instructions or administrationschedules for a patient suffering from a disease or condition (e.g., acancer, autoimmune disease, or cardiovascular disease) to use thepharmaceutical composition(s) containing an HSA linker, or any binding,diagnostic, and/or therapeutic agent conjugated thereto.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, methods,and kits of the present invention without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncover the modifications and variations of this invention provided theycome within the scope of the appended claims and their equivalents.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 Methods to Identify Residues in the HSA Linker forSite-Specific Conjugation of Cytotoxic or Cytostatic Drugs

To identify sites for specific conjugation of drug to HSA the crystalstructure is studied and surface exposed serine and threonine residuesare identified. These particular surface-exposed amino acids can then bemutated to cysteine allowing drug conjugation to the substitutedcysteine using a thiol-specific conjugating agent such as maleimide.Mild reduction may be required prior to drug conjugation. The number ofdrugs conjugated is controlled by the number of surface exposed cysteineresidues introduced into HSA. Serine and threonine are selected as themost suitable residues for mutation as they share the most structuralidentity with cysteine, however, other surface exposed residues may alsobe mutated to cysteine and successfully conjugated to cytostatic orcytotoxic drugs.

The crystal structure of HSA is deposited in the RSCB Protein Data Bank(1bm0-Sugio et al., “Crystal structure of human serum albumin at 2.5 Aresolution,” Protein Eng. 12:439-446 (1999)). This structure is analyzedusing the DeepView Swiss PDB Viewer downloaded from the Swiss Instituteof Bioinformatics. Serine and threonine residues with 50%, 40%, and 30%surface exposure were identified as the most suitable for mutation tocysteine (Table 2). Mutations can be introduced using standard molecularbiology procedures. Conjugation of a thiol reactive drug or chelatingagent to introduced cysteines can be tested using standard proteinchemistry techniques.

TABLE 2 % Surface Exposure Residue 50 T496 40 S58 30 T76, T79, T83,T125, T236, S270, S273, S304, S435, T478, T50

 T508

indicates data missing or illegible when filed

Example 2 Methods to Identify Residues in the HSA Linker forIntroduction of Asparagine-Linked Glycosylation Sites

To identify regions for introduction of asparagine-linked glycosylationsites in HSA, the crystal structure is studied to identify surfaceexposed (>30%) asparagine, serine and threonine residues that would besuitable for mutation. Glycosylation occurs on asparagine residues whenthe consensus sequence asparagine-x-serine/threonine is present, where xcannot be a proline. Table 2 lists possible mutation sites in HSA forthe introduction of asparagine-linked glycosylation.

TABLE 3 Residue Proposed Mutation Gln32 Asn Val46 Ser/Thr Asp56 AsnAsp63 Ser/Thr* Glu231 Asn Asp237 Asn Gln268 Asn Asp269 Ser/Thr Glu285Asn Ala320 Ser/Thr* Asp340 Asn Glu354 Asn Gln437 Asn Glu425 Asn Glu465Asn Asp494 Asn*

*These mutations have also been reported to occur very rarely in HSA(Carlson et al., “Alloalbuminemia in Sweden: Structural study andphenotypic distribution of nine albumin variants,” Proc. Nat. Acad. Sci.USA 89:8225-8229 (1992); Madison et al., “Genetic variants of humanserum albumin in Italy: point mutants and a carboxyl-terminal variant,”Proc. Nat. Acad. Sci. USA 91:6476-6480 (1994); Hutchinson et al., “TheN-terminal sequence of albumin Redhill, a variant of human serumalbumin,” FEBS Lett. 193:211-212 (1985); Brennan et al., “AlbuminRedhill (-1 Arg, 320 Ala-Thr): a glycoprotein variant of human serumalbumin whose precursor has an aberrant signal peptidase cleavage site,”Proc. Nat. Acad. Sci. USA 87:26-30 (1990); Minchiotti et al.,“Structural characterization of four genetic variants of human serumalbumin associated with alloalbuminemia in Italy,” Eur. J. Biochem.247:476-482 (1997); Peach et al., “Structural characterization of aglycoprotein variant of human serum albumin albumin Casebrook (494Asp-Asn),” Biochim. Biophys. Acta 1097:49-54 (1991)).

Example 3

B2B3-1 is a bispecific scFv antibody fusion molecule comprising B1D2, ahuman anti-ErbB2 scFv antibody (SEQ ID NO:27) and H3, a human anti-ErbB3scFv (SEQ ID NO:26). The two scFvs are joined by a modified human serumalbumin (HSA) linker. The anti-ErbB3 scFv, H3, is recombinantly fused tothe amino terminus of the HSA linker incorporating a short connectorpolypeptide and the anti-ErbB2 scFv, B1D2, is recombinantly fused to thecarboxy terminus of the modified HSA linker incorporating an additionalshort connector polypeptide. Each connector polypeptide is selectedbased on protease resistance properties. The modified HSA linkercontains two amino acid substitutions. A cysteine residue at position 34of native HSA is mutated to serine in order to reduce potential proteinheterogeneity due to oxidation at this site. An asparagine residue atamino acid 503 of native HSA, which in native HSA may be sensitive todeamidation which can result in decreased pharmacologic half-life, ismutated to glutamine. It is believed that B2B3-1 selectively binds ErbB2over-expressing tumors by virtue of its high affinity anti-ErbB2 scFvbinding moiety, which has a kD in the range of 10.0 nM to 0.01 nM andmore preferably a kD of about 0.3 nM. Subsequent binding of ErbB3 by theanti-ErbB3 scFv, which has a kD in the range of 50 to 1 nM and morepreferably about 16 nM, inhibits HRG induced phosphorylation of ErbB3.The modified HSA linker confers an extended circulating half-life on thebispecific molecule. B2B3-1 has a molecular weight of 119.6 kDa and ispreferably not glycosylated.

B2B3-1 inhibits ligand-induced phosphorylation of ErbB3 withsub-nanomolar potency; this activity is believed to be due, at least inpart, to the abundant expression of its dimerization partner, ErbB2,which facilitates specific targeting of cancer cells that express bothreceptors.

Example 4

As shown in FIG. 2, B2B3-1 variants inhibit HRG-induced pErbB3 in ZR75-1breast cancer cells. ZR75-1 breast cancer cells are treated with a doserange of B2B3-1 variants for 24 hours followed by HRG stimulation.pErbB3 levels are measured in cell lysates by ELISA and IC₅₀ values arecalculated together with the percent of inhibition. Shown are the meanIC₅₀ values (Y axis) with error bars representing replicate experiments.Percent inhibition values are shown above the corresponding bar.

ELISA Assays

Except as noted, ELISA reagents for total and phospho-ErbB3 ELISAs arepurchased from R&D Systems as DUOSET kits. 96-well NUNC MAXISORB platesare coated with 50 μl of an antibody and incubated overnight at roomtemperature. Next morning, plates are washed 3 times with 1000 μl/wellin a BIOTEK plate washer with Dulbecco's phosphate buffered salinewithout calcium or magnesium (PBS) with added Tween detergent (PBST)(0.05% Tween-20). Plates are subsequently blocked for about 1 hr at roomtemperature with 2% BSA in PBS. The plates are washed 3 times with 1000μl/well in the BIOTEK plate washer with PBST. Cells are grown at 37° C.and 5% carbon dioxide, washed with cold PBS, then harvested withmammalian protein extract (MPER) lysis buffer (Pierce, 78505) to which150 mM NaCl, 5 mM sodium pyrophosphate, 10 uM bpV (phen), 50 uMphenylarsine, 1 mM sodium orthovanadate, and protease inhibitor cocktail(Sigma, P2714) is added. 50 μL of cell lysates and standards diluted in50% Lysis buffer and 1% BSA are used in duplicates for furtherprocessing. Samples are incubated for 2 hrs at 4° C. on a plate shakerand washed as before. About 50 μl of a detection antibody diluted in 2%BSA, PBST is added and incubated for about 1-2 hrs at room temperature.For phospho-ErbB3, the detection antibody is directly conjugated tohorseradish peroxidase (HRP), while for total ErbB3 and biotinylatedmouse anti-human ErbB3 secondary detection antibody is used. The plateis washed as before. For total ErbB3 about 50 μl of Streptavidin-HRP isadded and incubation is for 30 min and the plates washed as before.About 50 μL of SUPERSIGNAL ELISA Pico (Thermo Scientific) substrate isadded and the plate is read using a FUSION plate reader. Duplicatesamples are averaged and, where shown, error bars represent the standarddeviation between the two replicates.

Example 5

Inhibition of phosphorylated ErbB3 (FIGS. 3A-D), AKT (FIGS. 4A-D), andERK (FIGS. 5A-D) following 24 hour pre-treatment with B2B3-1 variantsA5-HSA-B1D2 (panel A of FIGS. 3-5), H3-HSA-B1D2 (panel B of FIGS. 3-5),H3-HSA-F5B6H2 (panel C of FIGS. 3-5), and F4-HSA-F5B6H2 (panel D ofFIGS. 3-5) is measured. BT474 breast cancer cells are treated with adose range of B2B3-1 variants for 24 hours followed by HRG stimulation.pErbB3, pAKT, and pERK levels are measured in cell lysates by ELISA andIC₅₀ values are calculated together with the percent of inhibition.These results demonstrate that B1B2-1 is the only HSA linker conjugateof those tested that provides greater than 50% inhibition of HRG-inducedphosphorylation of AKT, ERK, and ErbB3 at a concentration of 10⁻⁸ molarand above.

Example 6

As shown in FIG. 6, treatment of BT474 breast tumor cells with B2B3-1variants causes G1 cell cycle arrest and a decrease in the population ofcells in S phase. BT474 cells are treated with 1 μM of B2B3-1 variantsand controls for 72 hours. After the end of treatment, cells aretrypsinized, gently resuspended in hypotonic solution containingpropidium iodide and single cells are analyzed by flow cytometry. Cellcycle distribution in G1 and S phases is measured using curve-fittingalgorithms designed for cell cycle analysis (FlowJo software cell cycleplatform).

Example 7

B2B3-1 (SEQ ID NO:16) inhibits ErbB3 activation, utilizing the abundantexpression of its dimerization partner, ErbB2, to target tumor cells. Ahigh affinity anti-ErbB2 scFv antibody, B1D2, facilitates targeting ofB2B3-1 to tumor cells over-expressing ErbB2. B1D2 is connected by amodified HSA linker to a lower affinity anti-ErbB3 scFv antibody, H3,which blocks binding of ErbB3's ligand, HRG. It is believed that theinhibition of ErbB3 phosphorylation and downstream AKT signalingmediated by B2B3-1 is due to this blockade. The ErbB2 binding scFv,B1D2, is derived from parent scFv C6.5, which possesses neitheragonistic nor antagonistic activity at ErbB2. B1D2, therefore, likelyfunctions solely as a targeting agent. The lower affinity binding of theErbB3 binding scFv is believed to prevent strong binding of B2B3-1 tonormal, non-cancerous tissues which express ErbB3 but little or noErbB2, thereby reducing the potential for non-specific toxicity. Intumor cells expressing both ErbB2 and ErbB3, there is an avidity effectof bispecific B2B3-1 binding to both receptors that overcomes the lowaffinity of the ErbB3 scFv allowing strong inhibition of HRG interactionwith ErbB3 receptor complexes.

The ability of B2B3-1 to inhibit HRG binding to ErbB3 is investigatedusing flow cytometry (FACS). Cells of the breast cancer cell line human(a variant of BT-474 that over-express ErbB2), are pretreated with 1 μMB2B3-1 then incubated with 10 nM biotinylated HRG 1β EGF domain. Afterextensive washing, binding is assessed using streptavidin-AlexaFluor 647conjugate. All incubations are performed at 4° C. FIG. 7 shows thatB2B3-1 is capable of blocking the binding of HRG to ErbB3. and appearsto provide 100% blockade at a concentration of 1 μM.

Example 8

After demonstrating B2B3-1's ability to block HRG binding to ErbB3, theeffect of B2B3-1 on ErbB3 signaling in vitro on two cell lines thatexpress ErbB3 and over-express ErbB2 is investigated. Human breastcancer cell lines BT-474-M3 (described in, e.g., Drummond et al. (2005)Clin. Cancer Res. 11:3392; Park et al. (2002) Clin. Cancer Res. 8:1172;Kirpotin et al. (2006) Cancer Res. 66:6732) and ZR7530 (obtainable fromthe US NIH Lawrence Berkeley National Laboratory breast cancer cellcollection) are serum starved overnight, pre-treated with a dosetitration of B2B3-1 for 24 hours and then stimulated for 10 minutes with5 nM of HRG 1β EGF domain. The phosphorylation status of ErbB3 and AKTis then examined using ELISA assays generally as described above. Theresults show that B2B3-1 inhibits HRG induced activation of both ErbB3and AKT phosphorylation in a dose-dependent manner and with potent IC₅₀sin both cell lines (FIGS. 8A-D).

Example 9

FIG. 9 shows the effect of B2B3-1 treatment on signaling proteins inBT474 breast cancer cells. Cells are treated with a dose range of B2B3-1for 24 hours, followed by heregulin stimulation. Levels of pErbB2,pErbB3, pErk and pAKT and their corresponding total protein levels aredetermined on cell lysates by Western blot analysis. Results indicatethat levels of at least pErbB2 and pErbB3 are reduced in adose-dependent manner by B2B3-1 treatment.

Example 10

FIG. 10 shows the immunoprecipitation-Western blot analysis of B2B3-1treated BT474 breast cancer cells. Cells are treated with a dose rangeof B2B3-1 for 24 hours, followed by heregulin stimulation.ErbB2-associated complexes are isolated from cell lysates using ananti-ErbB2 antibody followed by Western blot analysis to detect pErbB2and pErbB3 and the corresponding total protein levels. The results showthat B2B3-1 crosslinks ErbB2 to ErbB3, so that substantially more ErbB3and phospho-ErbB3 is precipitated by the anti-ErbB2 antibody.

Example 11

The anti-tumor activity of B2B3-1 is investigated in vitro using anumber of assays. In the first assay, the effect of B2B3-1 on theaccumulation of BT-474 or SKBR3 cells in G1 phase and the concomitantdecrease in S phase of the cell cycle is examined Briefly, cells aretreated with 1 μM B2B3-1 or PBS vehicle for 72 hours. After the end oftreatment, cells are trypsinized, gently resuspended in hypotonicsolution containing propidium iodide and single cells are analyzed byflow cytometry. Cell cycle distribution in G1 and S phases is measuredusing curve-fitting algorithms designed for cell cycle analysis (FlowJosoftware cell cycle platform, Tree Star, Inc.). B2B3-1 was found tomodestly decrease the percentage of cells in S phase and increase thepopulation of cells in G1 phase (FIG. 11A). In a second experiment, thenumber of cell colonies formed following treatment with B2B3-1 isstudied. BT-474 and SKBR3 breast cancer cells are plated in the presenceof 1 μM B2B3-1 and compared to cells plated in media only. Media only ormedia including treatment is replenished every 3 days. After 14 days thenumber of colonies is counted and compared to untreated cells. FIG. 11Billustrates the 40-45% decrease in the number of colonies formed whencells are treated with B2B3-1 compared to control cells. Finally, theability of B2B3-1 to inhibit cell proliferation is assessed in a cellimpedance assay using a Real-Time Cell Electronic Sensing System(RT-CES: ACEA Biosciences). BT-474 cells are seeded on plates integratedwith microelectronic sensor arrays and treated with a dose titration ofB2B3-1 or media only for 72 hours. Data reflecting the generation ofcell-electrode impedance response are collected every hour for 72 hoursand IC₅₀ values are calculated 68 hours after treatment. FIG. 11Cillustrates that B2B3-1 was able to inhibit impedance of BT-474 cellswith an IC₅₀ of 33 nM.

Example 12

We also investigated whether B2B3-1 exhibits agonistic activity based onits ability to simultaneously bind and cross-link ErbB2 and ErbB3receptors. Serum starved ZR75-1 breast cancer cells are incubated with 1μM B2B3-1 or PBS vehicle for 24 hours. Cells are also treated withB2B3-1 or PBS vehicle for 24 hours followed by a 10-minute stimulationwith 5 nM HRG 1β EGF domain. Cells are lysed and the pErbB3 content ofthe lysates is assessed by ELISA generally as described above. FIG. 12shows that cells treated with B2B3-1 alone contained levels ofphosphorylated ErbB3 that were comparable to the levels in untreatedcells, indicating that B2B3-1 does not act as an agonist promoting ErbB3phosphorylation.

Example 13

The ability of B2B3-1 to bind with specificity to ErbB2 and ErbB3 andnot to related ErbB family members, EGFR and ErbB4, is investigated byELISA. Plates are coated with the recombinant extracellular domain ofeither ErbB2 or ErbB3. Plates are blocked and incubated with ahalf-maximal binding concentration of B2B3-1 in the presence of adilution series of competing recombinant extracellular domains of EGFR,ErbB2, ErbB3 or ErbB4. The results show only soluble ErbB2 extracellulardomain blocked B2B3-1 binding to ErbB2-coated plates (FIG. 13A).Likewise, only soluble ErbB3 extracellular domain blocked B2B3-1 bindingto ErbB3-coated plates (FIG. 13B). These results are believed todemonstrate the specificity of the anti-ErbB2 arm B1D2 for ErbB2, and ofthe anti-ErbB3 arm H3 for ErbB3. The increased signal observed whensoluble ErbB2 extracellular domain was incubated with B2B3-1 on theErbB3 coated plate is assumed to be due to formation of an ErbB2, ErbB3,B2B3-1 complex on the plate.

Example 14

The ability of B2B3-1 to bind to tumor cells expressing both ErbB2 andErbB3 is studied using monospecific variants of B3B3-1. SKO-2 (SEQ IDNO:67) and SKO-3 (SEQ ID NO:68) are variants of B2B3-1 that lack theability to interact with ErbB2 or ErbB3, respectively.

SKO-2 and SKO-3 are constructed using the QUIKCHANGE Site DirectedMutagenesis kit (STRATAGENE) which uses oligonucleotide primer pairs,each complementary to opposite strands of the template vector. These areextended during temperature cycling generating a mutated plasmidcontaining staggered nicks. Following temperature cycling, the productis treated with Dpn I, which is used to digest the parental DNAtemplate. The nicked vector DNA containing the desired mutations is thentransformed into XL1-BLUE supercompetent cells STRATAGENE) to propogateplasmid DNA.

To create SKO-2, 5 nucleotides in the VH CDR3 loop of the anti-ErbB2scFv, B1D2, are mutated to create the following amino acidsubstitutions; H95A, W100hA, and E100jA. Mutations at these positionshave been demonstrated to knock-out binding of the B1D2 parent scFv,C6.5, to ErbB2 (Schier et al, 1996 JMB). Mutations are introduced to theB2B3-1 plasmid pMP9043 (SEQ ID NO:60) in a stepwise manner. First,mutations c295g and a296c are generated using primers 5′-GTA CTT TTG TGCCCG GGC CGA TGT GGG CTA CTG C-3′ (SEQ ID NO:61) and 5′-GCA GTA GCC CACATC GGC CCG GGC ACA AAA GTA C-3′ (SEQ ID NO:62) and temperature cyclingof 95° C. for 1 minute followed by 30 cycles of 95° C. for 1 minute, 55°C. for minute, and 65° C. for 17.2 minutes. Mutations are confirmed byDNA sequencing of plasmid DNA. To introduce mutations at t334g, g335c,and a341c, a second round of site-directed mutagenesis is performed onthe plasmid with sequence-confirmed mutations at c295g and a296c usingprimers 5′-GAC ATG TGC CAA GGC CCC CGC GTG GCT GGG AGT G-3′ (SEQ IDNO:63) and 5′-CAC TCC CAG CCA CGC GGG GGC CTT GGC ACA TGT C-3′ (SEQ IDNO:64) and temperature cycling of 95° C. for 30 seconds and 18 cycles of95° C. for 30 seconds, 55° C. for 1 minute and 68° C. for 17.2 minutes.Mutations are confirmed by DNA sequencing of resulting plasmid DNA.

To create SKO-3, the mutated B1D2 scFv is subcloned into the originalB2B3-1 plasmid to replace the anti-ErbB3 scFv, H3. Primers annealing toSKO-2, 5′ACAGTGGCGGCCGCCACCATGGGCTGGTCTCTGATCCTGCTGTTCCTGGTGGCCGTGGCCACGCGTGTGCTGTCCCAGGTGCAGCTCGTCCAGAGCGGCGC (SEQ ID NO:65) and5′GGAGGCGGCGCCCAGGACTGTCAGCTTGGTGCCACCGCCG (SEQ ID NO:66) are used toisolate the mutated B1D2 scFv from SKO-2 and to introduce Kas I and NotI restriction sites for subcloning into Kas I/Not I restriction digestedB2B3-1 plasmid. PCR is performed as follows: 94° C. for 1 minutefollowed by 30 cycles of 94° C. for 30 seconds, 58° C. for 1 minute, 72°C. for 1 minute, followed by one cycle of 72° C. for 5 minutes toamplify the mutant B1D2. Successful cloning is monitored by DNAsequencing. SKO-2 and SKO-3 plasmids are stably expressed from CHO-K1cells in shake flasks or 10 L WAVE bags and purified from conditionedmedia using Blue SEPHAROSE and cation exchange chromatography.

MALME-3M melanoma cells, which express approximately equal numbers ofErbB2 and ErbB3 receptors, are incubated with a dilution series ofB2B3-1, SKO-2, or SKO-3 in the presence of saturating concentrations ofa goat anti-HSA Alexafluor-647 conjugated antibody. Cell binding isassessed by flow cytometry and apparent binding affinities aredetermined for each molecule. Control cells are incubated with secondaryantibody alone. No measurable cell binding is observed for SKO-2, whichretains only the low affinity binding to ErbB3 mediated by H3 and lacksErbB2 binding activity. SKO-3, which retains a functional, high affinityErbB2 binding B1D2 scFv but lacks the ability to bind ErbB3 had a K_(D)of 6.7 nM. B2B3-1 bound cells with a K_(D) of 0.2 nM, demonstrating theincreased binding mediated by the bispecific design of this molecule(FIG. 14).

Example 15

The stability of B2B3-1 under physiological conditions is assessed byincubating 100 nM B2B3-1 in human, Cynomolgus monkey, or mouse serum at37° C. for a period of 120 hours. Samples are removed at 0, 24, 48, 72,96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3is measured by ELISA. This ELISA involves coating a 96-well plate withrecombinant ErbB2 extracellular domain overnight followed by a blockingstep and then incubation with a dilution series of B2B3-1. Plates arethen incubated with an Fc-ErbB3 extracellular domain fusion proteinfollowed by a goat antihuman-Fc-HRP conjugate. Plates are developed byaddition of supersignal chemiluminescense substrate. FIGS. 15A-C showthat B2B3-1 remains stable in serum from all three species underphysiological conditions, retaining comparable ability to bind bothErbB2 and ErbB3 at all time points measured.

Example 16 B2B3-1 Dose Response in BT-474-M3 Human Breast Cancer In VivoXenograft Model

The efficacy of B2B3-1 in vivo is assessed using nude mice bearing humanBT-474-M3 xenografts. Ten mice per group are treated with 12 doses of0.3, 1, 3, 10, 30 or 90 mg/kg of B2B3-1 every 3 days. Control groups areadministered PBS vehicle or HSA at an equimolar dose to the 90 mg/kgB2B3-1 dose. All doses are administered interperitoneally (i.p.). Tumorsize is measured twice a week and the corresponding tumor volume iscalculated. The results show that B2B3-1 treatment leads to significantreduction in BT-474-M3 tumor size as compared to the control group (FIG.16). Complete regressions were observed in each of the B2B3-1 treatmentgroups except mice treated with the lowest dose of B2B3-1 (0.1 mg/kg).

Example 17

As shown in FIGS. 17A-E, B2B3-1 reduces tumor size in multiple xenograftmodels in an ErbB2 dependent manner. B2B3-1 was efficacious in theCalu-3 (FIG. 17A), SKOV-3 (FIG. 17B), NCI-N87 (FIG. 17C), and MDA-MB-361(FIG. 17E) xenograft models expressing ErbB2 at >1×10⁵ receptors/cellbut worked less well in the ACHN (FIG. 17D) xenograft model whichexpresses 4.5×10⁴ ErbB2 receptors/cell. Mice were treated with 30 mg/kgof B2B3-1 every 3 days.

Example 18

Over-expression of ErbB2 converts B2B3-1 non-responder ADRr breastcancer xenograft model into a responder to B2B3-1 (FIGS. 18A and 18B).ErbB2 is over-expressed in ADRr breast cancer cells using a retroviralexpression system. Transfected cells expressing high levels of ErbB2(ADRr-E2) are selected using FACS and subsequently injectedsubcutaneously into nude mice to establish xenograft tumors. Mice aretreated with 30 mg/kg of B2B3-1 every 3 days. While no response toB2B3-1 was observed in wild type ADRr xenografts (FIG. 18A), ADRr-E2xenografts (FIG. 18B) responded to B2B3-1.

Example 19

As shown in FIGS. 19A-B, B2B3-1 activity correlates positively withErbB2 expression levels in vitro (FIG. 19A) and in vivo (FIG. 19B).B2B3-1 inhibition of ErbB3 phosphorylation is determined in 9 tumor celllines with expression levels of ErbB2 ranging from 5×10⁴ receptors/cellto 2.4×10⁶ receptors/cell using an ELISA assay. The extent of B2B3-1'sability to inhibit ErbB3 phosphorylation to basal levels (% pErbB3inhibition) was found to correlate positively with ErbB2 expressionlevels. Similarly, B2B3-1 activity is assessed in 10 tumor xenograftmodels expressing low to high levels of ErbB2. Xenograft response alsocorrelated positively with ErbB2 expression levels.

Example 20

B2B3-1 treatment of BT474-M3 breast tumor cells results in translocationof p27^(kip1) to the nucleus (FIG. 20A). BT474-M3 cells are treated with1 μM of B2B3-1 for 6 hours. The sub-cellular location of p27^(kip1) isassessed using immunofluorescence techniques. In cells treated withB2B3-1, p27^(kip1) translocated to the nucleus, which has been shown toresult in inhibition of cell proliferation. p27^(kip1) remained in thecytoplasm of untreated cells.

To further study the effect of B2B3-1 on the cell cycle, BT-474-M3 cellstreated with B2B3-1 for 72 hours are probed for the cell cycle regulatorCyclin D1 using Western blot analysis (FIG. 20B). The cytoskeletonprotein vinculin is used as a protein loading control in thisexperiment. B2B3-1 treatment resulted in a decrease in the levels ofCyclin D1 compared to untreated cells.

Example 21

As shown in FIGS. 21A-B, B2B3-1 treatment of BT474-M3 breast tumorxenografts results in translocation of p27^(kip1) to the nucleus. BT474breast tumor xenografts are treated with B2B3-1 (FIGS. 21A) at a dose of30 mg/kg or an equimolar dose of HSA (FIGS. 21B) every 3 days for atotal of 4 doses. Increased nuclear staining for p27^(kip1) was observedin B2B3-1 treated tumors compared to HSA control tumors indicating ananti-proliferative effect of B2B3-1 in vivo.

Example 22

B2B3-1 treatment results in a reduction of the proliferation marker Ki67in BT474 breast cancer xenograft tumors. BT474-M3 breast tumorxenografts are treated with B2B3-1 (FIG. 22A) at a dose of 30 mg/kg oran equimolar dose of HSA (FIG. 22B) every 3 days for a total of 4 doses.Subsequent staining of tumor sections for Ki67 demonstrated a reducedexpression pattern for B2B3-1 treated tumors compared to HSA treatedtumors.

Example 23

B2B3-1 treatment results in a reduction of vessel density in BT474-M3breast cancer xenograft tumors, as determined by assaying for CD31expression (FIGS. 23A-B). BT474 breast tumor xenografts are treated withB2B3-1 (FIG. 23A) at a dose of 30 mg/kg or an equimolar dose of HSA(FIG. 23B) every 3 days for a total of 4 doses. Tumors are stained forthe presence of vascular marker CD31. Tumors treated with B2B3-1 show amarked decrease in vessel density compared to control tumors treatedwith HSA.

Example 24 B2B3-1 Inhibits Phosphorylation of ErbB3 In Vivo

BT-474-M3 xenograft tumors are treated with 30 mg/kg B2B3-1 or 17.5mg/kg HSA every 3 days for a total of 4 doses and tumors are harvested24 hours after the final dose. Tumors are lysed and subjected toSDS-PAGE followed by Western analysis to assess relative levels ofphosphorylation of B2B3-1's target ErbB3. Equal quantities of proteinare loaded in each lane and total protein levels are controlled byprobing for beta tubulin. Western blot analysis using antibodiesspecific for phosphorylated ErbB3 show that B2B3-1 treated tumorscontain less pErbB3 than HSA treated tumors (FIG. 24A). Densitometry ofthe western blot analysis followed by normalization of the mean pErbB3integral band intensity to the mean beta tubulin integral band intensitydemonstrated that B2B3-1 treated tumors contained significantly lesspErbB3 than control HSA treated tumors (FIG. 24B). These data confirmedthat B2B3-1 inhibits phosphorylation of its target ErbB3 in vivo.

Example 25 In Vivo Activity of B2B3-1 in BT-474-M3 Xenografts which haveReduced PTEN Activity

ShRNA technology is applied to knock out the activity of the tumorsuppressor gene phosphatase and tensin homolog (PTEN) in BT-474-M3breast cancer cells. Briefly, cultured BT-474-M3 cells are transfectedwith shPTEN or shControl RNA by retroviral transfection. Transfectedcells with reduced PTEN are selected using FACS and subsequentlyinjected subcutaneously into the right flank of nude mice to establishxenograft tumors. Cells transfected with a control vector are injectedinto the left flank of the same mouse. Mice are treated with 30 mg/kgB2B3-1 every 3 days or 10 mg/kg trastuzumab every week. HSA is injectedas a control at an equimolar dose to B2B3-1. All injections are donei.p.

B2B3-1 and trastuzumab promoted a reduction in the size of tumors formedby control BT-474-M3 breast cancer cells (FIG. 25A), whereas only B2B3-1(and not trastuzumab) promoted a reduction in the size of tumors formedby BT-474-M3 human breast cancer cells lacking expression of PTEN (FIG.25B).

Example 26 B2B3-1 Inhibits ErbB3 Signaling in BT-474-M3 Breast CancerCells Having Reduced PTEN Activity

The ability of B2B3-1 to inhibit phosphorylation of ErbB3 signaling intumor xenografts is studied using the PTEN deficient BT-474-M3 modeldescribed above. Xenograft tumors of the engineered cell line or controlcell line are treated with 30 mg/kg B2B3-1, 17.5 mg/kg HSA every 3 daysor 10 mg/kg trastuzumab weekly and tumors are harvested 24 hours afterthe final dose. Tumors are lysed and subjected to SDS-PAGE followed byWestern analysis to assess relative levels of phosphorylation ofB2B3-1's target ErbB3, AKT and total PTEN levels. Equal quantities ofprotein are loaded in each lane and total protein levels are controlledby probing for PCNA. Western blot analysis using antibodies specific forphosphorylated ErbB3 shows that B2B3-1 treated tumors contain less pAKTthan HSA treated or Herceptin treated tumors (FIG. 26A). Densitometry ofthe western blot analysis followed by normalization of the mean pAKTintegral band intensity to the mean PCNA integral band intensitydemonstrated that B2B3-1 treated tumors contained significantly lesspAKT than control HSA treated and Herceptin-treated tumors (FIG. 26B).

Example 27

The pharmacokinetic parameters for B2B3-1 are investigated in nu/numice. Animals are randomized into groups and administered intravenous(IV) with a single dose of 5, 15, 30, or 45 mg/kg B2B3-1 (FIGS. 27A-D,respectively). Blood is collected pre-dose and at 0.5, 4, 8, 24, 48, 72,and 120 hours post dose. Three mice are used for each time point. Serumlevels of B2B3-1 are measured using two ELISA methods. The first methodrequires functional binding of B2B3-1 to both ErbB2 and ErbB3 while thesecond method measures only the HSA component of B2B3-1 in the serum.The HSA ELISA utilizes a polyclonal-anti HSA capture antibody and aHRP-conjugated polyclonal anti-HSA detection antibody. A reduction inB2B3-1 serum concentration measured using the ErbB2/ErbB3 binding methodversus the HSA method would indicate a loss in functional B2B3-1. FIGS.27A-D show that the pharmacokinetic properties of B2B3-1 are comparablewhen assessed using either ELISA method, indicating that B2B3-1 isstable in circulation in mice.

Example 28

B2B3-1 serum concentrations are fit using a two-compartment,biexponential model and show biphasic disposition. Terminal half-liveswere calculated to be 17, 16, 23, and 18 hrs for the 5, 15, 30, or 45mg/kg doses, respectively, and are shown in Table 4. Increases in B2B3-1dose resulted in a linear increase in exposure (FIG. 28).

TABLE 4 Pharmacokinetic properties of B2B3-1 in mice and Cynomolgusmonkeys. Dose T½β AUC Clearance (mg/kg) Species N (hrs) (hr-μg/ml)(ml/hr/kg) 5 Mouse (single dose) 3 16.9 1.58E+03 3.19 15 Mouse (singledose) 3 16.2 6.10E+03 2.47 30 Mouse (single dose) 3 22.6 1.18E+04 2.5445 Mouse (single dose) 3 17.5 1.84E+04 2.46 4 Cynomolgus 2 39.1 3.44E+031.17 monkey (1st dose) 4 Cynomolgus 2 44.9 7.20E+03 0.60 monkey (4thdose) 20 Cynomolgus 2 33.1 2.29E+04 0.88 monkey (1st dose) 20 Cynomolgus2 122.5 8.20E+04 0.25 monkey (4th dose) 200 Cynomolgus 4 68.8 3.18E+050.64 monkey (1st dose) 200 Cynomolgus 2 69.7 5.72E+05 0.35 monkey (4thdose) 200 Cynomolgus 2 66.6 5.99E+05 0.34 monkey (4th dose)* *recoveryanimals

Example 29

Blood samples for pharmacokinetic analysis are also obtained from a doserange-finding toxicology study in female Cynomolgus monkeys. In thisstudy, animals are infused with 4, 20 or 200 mg/kg of B2B3-1administered every 3 days for 4 doses. Sampling occurred prior to and 5minutes after dosing on each dosing day (study days 1, 4, 7 and 10) toprovide pre-dose and peak/trough concentrations, and at 1, 2, 4, 8, 24and 48 hours after the end of the first infusion on day 1 and at 1, 2,4, 8, 24, 48, 72 and 120 hours after the last infusion on day 10. Forrecovery animals dosed at 200 mg/kg serum samples are also collected at168, 336 and 456 hours after the last infusion.

Cynomolgus monkey serum samples are assayed using the ErbB2/ErbB3 ELISAmethod described previously. Serum concentrations for each dose over thetime course are shown in FIG. 29. The analysis shows that meanconcentration-time profiles for serum B2B3-1 after dosing on days 1 and10 were qualitatively similar with concentrations generally decliningwith time from Cmax. Mean half-life estimates ranged from 38.3-67.2hours on day 1 and 45.0 to 121.0 hours on day 10 (Table 4).

Example 30

The plasmid encoding the B2B3-1 bispecific scFv antibody fusion proteinis created combining gene sequences of a unique human anti-ErbB3 scFv(designated “H3”), a human anti-ErbB2 scFv (designated “B1D2”), and amodified human serum albumin (HSA) linker. The anti-ErbB3 scFv, H3, isrecombinantly linked to the amino terminus of the HSA linker via aconnecting peptide (Ala-Ala-Ser) and the anti-ErbB2 scFv, B1D2, isgenetically linked the carboxy terminus of the HSA linker via aconnecting peptide (Ala-Ala-Ala-Leu—SEQ ID NO:5). The peptide connectorsare formed through the introduction of restriction sites duringconstruction of the mammalian expression vector and are synthesized withoptimized codon usage for mammalian expression together with the singlechain antibody fragments and HSA linker.

The B1D2 scFv is selected from a combinatorial phage display librarycreated by mutagenesis of the ErbB2-binding scFv C6.5, which is selectedfrom a non-immune phage display library. The H3 scFv is selected from anon-immune phage display library originally made by Sheets et al. Thegene sequences encoding the B1D2 and H3 single chain antibody fragmentsare optimized for CHO cell codon preferences and synthesized forsubsequent construction of the B2B3-1 encoding plasmid.

The modified HSA linker contains two amino acid substitutions. Acysteine residue at position 34 is mutated to serine in order to reducepotential protein heterogeneity due to oxidation at this site. Anasparagine residue at amino acid 503 is mutated to glutamine, which inwild type HSA is sensitive to deamination and can result in decreasedpharmacologic half-life.

The gene sequence encoding the modified HSA linker is synthesized withoptimized codon usage for mammalian expression for subsequentconstruction of the B2B3-1 encoding plasmid.

Example 31

The B2B3-1 coding sequence is cloned into pMP10k base vector usingstandard molecular biology techniques to create plasmidpMP10k4H3-mHSA-B1D2, shown in FIG. 30. For the most part this constructemploys commonly used genetic elements. B2B3-1 expression is driven bythe human GAPD promoter. This vector utilizes genetic elements referredto as Matrix Attachment Regions or MAR elements. The MAR geneticelements control the dynamic organization of chromatin, and insulatenearby genes from the effect of surrounding chromatin thereby increasingcopy number dependent, position-independent, expression of genes. MARelements have been shown to improve the probability of isolating a cloneexhibiting the desired level of expression for the production of arecombinant protein and to increase the stability of production. The MARelements used in the B2B3-1 constructs are non-coding human MARelements. In addition to the B2B3-1 plasmid, a neomycin antibioticresistance plasmid (FIG. 31) and a hygromycin resistance plasmid (FIG.32) are also used to select for stable transformants.

Example 32 First Round of Gene Transfection

Chinese Hamster Ovary CHO-K1 cells are purchased from ATCC (ATCC #CCL-61). The CHO-K1 cell line is a serum and proline dependent adherentsub-clone of the parental CHO cell line created by T. T. Puck. TheCHO-K1 cells used for B2B3-1 transfection are pre-adapted for suspensiongrowth in serum free media prior to transfection. An iterativetransfection procedure is used to develop the B2B3-1 cell line.Twenty-four hours before transfection, CHO-K1 cells are sub passaged to1.0×10⁶ cells/mL in SFM4CHO (Serum Free) medium (HyClone, Logan, Utah)supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and0.016 mM thymidine. On the day of transfection, cells are resuspended inOptiMEM medium (Invitrogen Corp, Carlsbad, Calif.) and 40,000 cells areplaced in each well of a twenty-four well plate. In the firsttransfection, the B2B3-1 expression plasmid (pMP10k4H3-mHSA-B1D2) andthe neomycin resistance plasmid (FIG. 30; pSV2-neo (Selexis, Inc.,Marlborough, Mass.) are mixed together using a molar plasmid ratio of75:1 (B2B3-1:neomycin resistance). The plasmid mixture is subsequentlymixed with a cationic lipid transfection reagent (Lipofectamine LTX,Invitrogen Corp, Carlsbad, Calif.) and lipid/DNA complexes are allowedto form for thirty minutes. The DNA/Lipid complex is then added to theCHO-K1 cells and the 24-well plates are placed in a 37° C., 5% CO₂incubator.

Example 33 Selection and Screening for High Producers

The contents of each transfection well are washed with PBS, trypsinizedand distributed across two, ninety-six well plates. The growth mediaused consists of DMEM/F12 (Invitrogen Corp, Carlsbad, Calif.) with 10%FBS (Invitrogen Corp, Carlsbad, Calif.) and 500 mg/L of geneticin (G418;Invitrogen Corp, Carlsbad, Calif.). Media in the 96-well plates ischanged on day 4 to SFM4CHO medium supplemented with 8 mM L-glutamine,0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 500 mg/L geneticin.Following an additional two weeks of culture in selection medium,surviving cells have formed well-defined colonies. The clones areevaluated using quantitative spot blot techniques. The top producingcolonies are trypsinized, and expanded to a single well of a 24-wellplate.

A seven day productivity assay is used to screen for high B2B3-1producing colonies. Upon expansion the cells in 24-well plates areallowed to proliferate for seven days in SFM4CHO medium supplementedwith 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mMthymidine. The B2B3-1 concentration in the spent media is determined Topclones from the 24-well scale are expanded into 125 mL baffled shakeflasks. A seven day study in the shake flask in SFM4CHO mediumsupplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and0.016 mM thymidine is used to screen the cell pools for growth andB2B3-1 production.

Example 34 Second Round of Gene Transfection

The highest producing cell pool determined from the first round oftransfection (supra) is transfected a second time to increaseproduction. Twenty-four hours before transfection, the cell pool is subpassaged to 1.0×10⁶ cells/mL in SFM4CHO (Serum Free) medium supplementedwith 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mMthymidine. On the day of transfection, cells are resuspended in OptiMEMmedium (Invitrogen Corp, Carlsbad, Calif.) and 40,000 cells re placed ineach well of a twenty-four well plate. In the first transfection, theB2B3-1 and hygromycin resistance plasmid (FIG. 32; pTK-Hyg (Clontech,Mountain View, Calif.)) are mixed together using a molar plasmid ratioof 50:1 (B2B3-1:hygromycin resistance). The plasmid mixture issubsequently mixed with a cationic lipid transfection reagent(Lipofectamine LTX, Invitrogen Corp) and lipid/DNA complexes are allowedto form for thirty minutes. The DNA/Lipid complex is then added to thecell pool and the 24-well plates are placed in a 37° C., 5% CO₂incubator.

Example 35 Selection and Screening for High Producers from SecondTransfection

The contents of each transfection well are washed with PBS, trypsinizedand distributed across two, 96-well plates. The growth media usedconsists of DMEM/F12 supplemented with 10% FBS and 400 mg/L ofhygromycin B (Invitrogen Corp). Media in the 96-well plates is changedon day 4 to Hyclone SFM4CHO medium supplemented with 8 mM L-glutamine,0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 400 mg/L ofhygromycin B. After an additional two weeks of selection, survivingcells have formed well-defined colonies. The clones are evaluated usingquantitative spot blot techniques. The top producing colonies aretrypsinized, and expanded to a single well of a 24-well plate.

A seven day productivity assay is used to screen for high B2B3-1producing colonies.

Upon expansion the cells are allowed to proliferate for seven days, andthe B2B3-1 concentration in the spent media is determined.

Top clones from the 24-well plates are expanded into 125 mL baffledshaker flasks in the Hyclone SFM4CHO medium supplemented with 8 mML-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. A sevenday study in shake flask is used to screen the cell pools for growth andB2B3-1 production. The spent media is quantitated using Protein Aresinand an HPLC instrument.

Example 36 Limiting Dilution Cloning

The best growing and highest B2B3-1-producing colony identified by theproductivity assay is transferred from the 125 mL shaker flask andplated in five 96-well plates at a cell concentration calculated to giveone cell/well. The 96-well plates are placed in an incubator at 37° C.and 5% CO₂. The wells are examined bi-weekly to track formation ofcolonies. Colonies arising from a single cell are identified based onthe symmetrical shape of the colony. Wells containing such colonies aremarked for further screening by 24-well 7-day assessment, and 125 mLshaker flask 7-day assessment.

The second round of limiting dilution cloning is performed in a similarmanner to the first round. An additional 100 mL fed batch evaluation isperformed to confirm clone choice. A pre-seed bank is cryopreserved.

Example 37 Formulation of B2B3-1

B2B3-1 is administered once a week via intravenous infusion over aperiod of 60 or 90 minutes, depending on patient tolerability. B2B3-1 isformulated in a sterile 20 mM L-histidine hydrochloride, 150 mM sodiumchloride, pH 6.5 solution at a concentration of 25 mg/mL foradministration to a patient (e.g., a human).

Example 38 Treatment of Breast Cancer

If a patient's cancer is believed to be expressing high levels ofepidermal growth factor receptors, including ErbB2 (HER2/neu), thentreatment with an HSA linker conjoined to an ErbB2 biding moiety, suchas B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9,or B3B3-10 (see Table 6, below) would be indicated. Such would be thecase where genotypic or histologic screens of cancer biopsies revealsincreased expression of ErbB2 in the patient's tumor.

A B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO:16) is administeredto a patient diagnosed with breast cancer once a week or twice a weekvia intravenous infusion over a period of, e.g., 60 or 90 minutes,depending on patient tolerability, at a dose no higher than 30 mg/kg.The B2B3 HSA linker conjugate is formulated in a sterile 20 mML-histidine hydrochloride, 150 mM sodium chloride, pH 6.5 solution at aconcentration of 25 mg/mL for administration to the patient. A cliniciansupervising the administration of the B2B3 HSA linker conjugate followscommon formulation and dosing practices to determine the proper courseof therapy for the patient.

The clinician may further co-administer one or more therapies with theB2B3 HSA linker conjugate. E.g., one or more therapeutic drugs orcompounds may be administered in combination with the B2B3 HSA linkerconjugate, such as the common chemotherapeutic regimen for the treatmentof breast cancer, which includes doxorubicin, cyclophosphamide, andpaclitaxel. Alternatively, a clinician can administer the B2B3 HSAlinker conjugate in combination with surgical or radiation therapy totreat breast cancer the patient.

Example 39 Treatment of Ovarian Cancer

If a patient's cancer is believed to be expressing high levels ofepidermal growth factor receptors, including ErbB2 (HER2/neu), thentreatment with an HSA linker conjoined to an ErbB2 biding moiety, suchas B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9,or B3B3-10 (see Table 6, below) would be indicated. Such would be thecase where genotypic or histologic screens of cancer biopsies revealsincreased expression of ErbB2 in the patient's tumor.

A B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO:16) is administeredto the patient diagnosed with ovarian cancer alone or in combinationwith one or more other therapies essentially as described in thepreceding Example.

Example 40 Additional HSA Linker Conjugates

HSA linker conjugates are constructed using one or more of the elements(groups A-E) listed in Table 5 below. In particular, an HSA linkerconjugate, which is shown as Group C in Table 5 below, incorporates oneor more binding moieties selected from groups A and E shown in Table 5.In addition, the HSA linker conjugates can also include one or morepeptide connectors, which are selected from groups B and D in Table 5,at each of the amino and carboxy terminal ends of the HSA linker.Peptide connectors can be repeated or truncated to increase or decreasethe length of the connector sequence.

Example 41 In Vivo, B2B3-1 Dosed q7d Shows Equivalent Efficacy as B2B3-1Dosed q3d

B2B3-1 efficacy using a q7d (once every 7 days) dosing regimen isdetermined in in female athymic nude mice (nu/nu) from Charles RiverLabs, 5-6 weeks of age bearing xenograft tumors of the human breastcancer cell line BT-474-M3 (FIG. 33). Mice receive a subcutaneousestrogen-releasing implant in the opposite flank (0.72 mg pellet, 60day, slow-release, Innovative Research of America, Sarasota, Fla.) 24 hprior to the injection of 20×10⁶ human BT-474-M3 cells in PBS. Dosing isinitiated when tumor growth is established (tumor volumes ofapproximately 400 mm3) and B2B3-1 is administered to 10 mice per groupeither once every 3 days (q3d) at 30 mg/kg for the course of the studyor once every 7 days at 22 mg/kg, 66 mg/kg, 132 mg/kg or 198 mg/kg byintraperitoneal injection. Tumors are measured twice a week usingdigital calipers. Tumor volume is calculated using the formula:π/6×(W²×L), where W is the short diameter and L is the long diameter.Pharmacokinetic calculations suggest that a 66 mg/kg dose q7d shouldgive a similar exposure of B2B3-1 to the xenograft tumor as a 30 mg/kgdose q3d. PBS vehicle is used as a negative control. B2B3-1 efficacy wasequivalent for the 30 mg/kg, q3d dose and the 3 highest doses of B2B3-1administered q7d, indicating a q7d dosing schedule for B2B3-1 issuitable in this model.

Example 42 B2B3-1 and Trastuzumab have Different Mechanisms of ErbB3Inhibition

The ability of B2B3-1 to inhibit heregulin induced ErbB3 activity istested using Western blot analysis. Monolayers of serum-starvedBT-474-M3 cells are treated for 24 hours with 100 nM B2B3-1 ortrastuzumab and then stimulated with 5 nM HRG 1β EGF for 10 minutes.Cells are also treated with 10nM and 100 nM B2B3-1 or 10nM and 100 nMtrastuzumab and left unstimulated. Lysates are subjected to immunoblotanalysis for ErbB3, pErbB3, AKT, and pAKT. Western blot analysis (FIG.34) demonstrated that B2B3-1 treatment results in inhibition of pErbB3and pAKT in a ligand dependent manner, whereas inhibition of pErbB3 andpAKT by trastuzumab was only seen in the absence of ligand.

Example 43 B2B3-1 has an Additive Effect when Administered withTrastuzumab In Vitro

The effects of B2B3-1, trastuzumab and the combination of both drugs onthe growth of cancer cell spheroids was examined using four differentbreast cancer cell lines. 2,000 cells of BT-474-M3, SKBR3 (ATCC), orMDA-MB-361 (ATCC) human breast cancer cells were seeded in round-bottomlow adherence 96-well plates (Corning® 96 Well Clear Round Bottom UltraLow Attachment Microplate-Product #7007) and the following day thespheroids were measured and treated with a dose range of B2B3-1,trastuzumab or a combination of both at a ratio of 3 fold molar excessB2B3-1 to trastuzumab. After 12 days of growth the surface area of thespheroids was measured and compared to untreated cells. As can be seenin FIGS. 35A-C, the combination of B2B3-1 with trastuzumab over a rangeof concentrations resulted in greater inhibition of spheroid growthcompared to the single agents in all cell lines tested at all but thelowest concentrations of drug(s). These results also indicate thatB2B3-1 does not compete with trastuzumab for binding to ErbB2 (HER2).

Example 44 B2B3-1 has an Additive Effect when Administered withTrastuzumab In Vivo

The effects of B2B3-1 when co-administered with trastuzumab in vivo isstudied in female athymic nude mice (nu/nu) from Charles River Labs, 5-6weeks of age, using a BT-474-M3 xenograft model. Mice receive asubcutaneous estrogen-releasing implant in the opposite flank (0.72 mgpellet, 60 day, slow-release, Innovative Research of America, Sarasota,Fla.) 24 h prior to the injection of 20×10⁶ human BT-474-M3 cells inPBS. Dosing is initiated when tumor growth is established (tumor volumesof approximately 400 mm³) Tumors are measured twice a week using digitalcalipers. Tumor volume is calculated using the formula: π/6×(W²×L),where W is the short diameter and L is the long diameter. Ten mice pergroup are administered B2B3-1 at 3 mg/kg or 10 mg/kg q3d, trastuzumab at1 mg/kg or 0.1 mg/kg q7d or the combination of both drugs for the courseof the study by intraperitoneal injection. All combinations of B2B3-1and trastuzumab (10 mg/kg B2B3-1+1 mg/kg trastuzumab, 10 mg/kgB2B3-1+0.1 mg/kg trastuzumab, 3 mg/kg B2B3-1+1 mg/kg trastuzumab, 3mg/kg B2B3-1+0.1 mg/kg trastuzumab) are dosed as for the correspondingsingle agent.

As shown in FIG. 36, substantially greater efficacy was seen for all thecombinations compared to the single agents and significant efficacy wasobserved from at least as early as day 20 onward for the combinationgroups dosed with 10 mg/kg B2B3-1 and both doses of trastuzumab and with3 mg/kg B2B3-1 and 1 mg/kg trastuzumab. In the 10 mg/kg B2B3-1+1 mg/kgtrastuzumab combination group, 5 out of the 10 mice had completelyregressed tumors compared to 0 out of 10 for the single agent groupsgiven equivalent doses as the combination. In the 3 mg/kg B2B3-1+1 mg/kgtrastuzumab combination group, 7 out of 10 mice had completely regressedtumors compared to 0 out of 10 for the single agent groups givenequivalent doses as the combination. These results indicate that B2B3-1does not compete with trastuzumab for binding to ErbB2 (HER2). Theseresults also demonstrate that treatment with a combination of at least 3mg/kg of B2B3-1 and at least 0.1 mg/kg trastuzumab is more effectivethan treatment with 3 mg/kg or 10 mg/kg of B2B3-1 alone or with 0.1mg/kg or 1 mg/kg of trastuzumab alone. In particular, the combination ofat least 3 mg/kg of B2B3-1 and 1 mg/kg trastuzumab induces essentiallycomplete tumor regression in at least about 50% of nude mice carryinghuman breast tumor cell xenografts, while the same concentrations ofeither B2B3-1 or trastuzumab alone do not provide complete regression ineven 10% of such mice.

Further provided are specific embodiments of the HSA linkers, peptideconnectors, and binding moieties discussed above. Table 6, below, liststen HSA linker conjugates with varying ErbB2-specific or ErbB3-specificbinding moieties, as well as peptide connectors, at the amino andcarboxy termini of an HSA linker.

Those skilled in the art will recognize, and will be able to ascertainand implement using no more than routine experimentation, manyequivalents of the specific embodiments described herein. Suchequivalents are intended to be encompassed by the following claims. Anycombinations of the embodiments disclosed in the dependent claims arecontemplated to be within the scope of the disclosure.

The disclosure of each and every US and foreign patent and pendingpatent application and publication referred to herein is herebyincorporated herein by reference in its entirety.

TABLE 6 HSA Linker Amino Terminal N-Terminal C-Terminal Carboxy TerminalConjugate Binding Moiety Connector HSA Connector Binding Moiety B2B3-1H3 (SEQ ID NO: 26) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27)(SEQ ID NO: 16) (SEQ ID NO: 5) B2B3-2 A5 (SEQ ID NO: 28) AASmHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 17)(SEQ ID NO: 5) B2B3-3 A5 (SEQ ID NO: 28) AAS mHSA (SEQ ID NO: 1) AAALF5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 18) (SEQ ID NO: 5) B2B3-4A5 (SEQ ID NO: 28) AAS mHSA (SEQ ID NO: 1) AAAL ML3.9 (SEQ ID NO: 29)(SEQ ID NO: 19) (SEQ ID NO: 5) B2B3-5 B12 (SEQ ID NO: 30) AASmHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 20)(SEQ ID NO: 5) B2B3-6 B12 (SEQ ID NO: 30) AAS mHSA (SEQ ID NO: 1) AAALF5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 21) (SEQ ID NO: 5) B2B3-7F4 (SEQ ID NO: 31) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27)(SEQ ID NO: 22) (SEQ ID NO: 5) B2B3-8 F4 (SEQ ID NO: 31) AASmHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 23)(SEQ ID NO: 5) B2B3-9 H3 (SEQ ID NO: 26) AAS HSA (SEQ ID NO: 3) AAALB1D2 (SEQ ID NO: 27) (SEQ ID NO: 24) (SEQ ID NO: 5) B2B3-10H3 (SEQ ID NO: 26) AAS mHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32)(SEQ ID NO: 25) (SEQ ID NO: 5)

APPENDIX 1 SEQUENCES, SEQUENCE ANNOTATIONS AND SEQUENCE ALIGNMENTSpMP9043 SEQ ID NO: 60gtgccgacgatagagcagacctcgctaaatatatctgcgagaatcaggattccattagctctaagctgaaagaatgttgcgagaagcccctcctggaaaagagtcattgtatcgccgaggtggaaaacgacgagatgccagcagatctgccatcactcgctgccgactttgtggaatccaaagatgtctgcaagaattacgcagaggctaaagacgtgttcctggggatgtttctgtatgagtacgcccggcgtcaccccgattatagcgtcgtgctcctgctccgactggcaaagacctacgaaacaactctggagaaatgttgcgctgccgcagaccctcatgaatgttatgctaaggtgttcgatgagtttaagccactcgtcgaagagccccagaacctgattaaacagaattgcgaactgttcgagcagctcggtgaatacaagtttcagaacgccctgctcgtgcgttataccaaaaaggtccctcaggtgtctacaccaactctggtggaggtcagtaggaatctgggcaaagtgggatcaaagtgttgcaaacaccccgaggcaaagagaatgccttgtgctgaagattacctctccgtcgtgctgaaccagctctgcgtgctgcatgaaaagaccccagtcagcgatcgggtgacaaaatgttgcaccgaatctctggtcaatcgccgaccctgtttcagtgccctcgaagtggacgaaacttatgtgcctaaggagtttcaggctgaaacattcacctttcacgccgatatctgcactctgtccgagaaagaaaggcagattaagaaacagacagcactggtcgagctcgtgaagcataaaccaaaggctaccaaggagcagctgaaagccgtcatggacgatttcgcagcttttgtggaaaagtgttgcaaagccgacgataaggagacttgtttcgcagaagaggggaaaaagctcgtggctgccagccaggcagctctgggtctggccgcagctctgcaggtgcagctcgtccagagcggcgctgaggtgaagaagccaggcgagtccctgaagatctcctgtaagggctccggctacagcttcacctcctactggatcgcttgggtgaggcagatgccaggaaagggactggagtacatgggcctgatctaccctggcgactccgacaccaagtactccccatccttccagggccaggtgaccatcagcgtggacaagtccgtgtctaccgcctacctgcaatggtcctccctgaagccttctgactctgccgtgtacttttgtgcccggcacgatgtgggctactgcaccgaccggacatgtgccaagtggcccgagtggctgggagtgtggggacagggaacactggtgacagtgagttctggcggtggcggctcttccggcggtggctctggtggcggcggatctcagagcgtgctgacacagccacctagcgtgtccgctgcccctggccagaaggtgacaatcagctgctccggcagctcttccaacatcggcaacaactacgtgtcttggtatcagcagctgcccggaacagctccaaaactgctgatctatgaccacaccaatcggcctgccggcgtgccagatcggttctctggctctaagagcggcacctccgccagcctggctatctctggcttcagatctgaggatgaggctgactactattgtgcctcctgggactacaccctgtctggctgggtgttcggcggtggcaccaagctgacagtcctgggatgatgactcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctccUtcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggtccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtacctagaagttcctattccgaagttcctattctctagaaagtataggaacttccttggccaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatagcacgtactacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttctagaattcgctgtctgcgagggccagctgttggggtgagtactccctctcaaaagcgggcatgacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcgtccatctggtcagaaaagacaatctttttgttgtcaagcttgaggtgtggcaggcttgagatctggccatacacttgagtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaactgcagatatccagcacagtggcggccgccaccatgggctggtctctgatcctgctgttcctggtggccgtggccacgcgtgtgctgtcccaggtgcagctgcaggagtctggcggcggactggtgaagcctggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctcctcctactggatgtcctgggtgcggcaggcccctggcaagggcctggagtgggtggccaacatcaaccgggacggctccgcctcctactacgtggactccgtgaagggccggttcaccatctcccgggacgacgccaagaactccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgccagggaccggggcgtgggctacttcgacctgtggggcaggggcaccctggtgaccgtgtcctccgctagtactggcggcggaggatctggcggaggagggagcgggggcggtggatcccagtccgccctgacccagcctgcctccgtgtccggctcccctggccagtccatcaccatcagctgcaccggcacctcctccgacgtgggcggctacaacttcgtgtcctggtatcagcagcaccccggcaaggcccctaagctgatgatctacgacgtgtccgaccggccttccggcgtgtccgacaggttctccggctccaagtccggcaacaccgcctccctgatcatcagcggcctgcaggcagacgacgaggccgactactactgctcctcctacggctcctcctccacccacgtgatctttggcggcggaacaaaggtgaccgtgctgggcgccgcctccgacgctcacaagagcgaagtggcacataggttcaaagatctgggcgaagagaactttaaggccctcgtcctgatcgctttcgcacagtacctccagcagtctccctttgaagatcacgtgaaactggtcaatgaggtgaccgaatttgccaagacatgcgtggctgatgagagtgcagaaaactgtgacaaatcactgcatactctctttggagataagctgtgcaccgtcgccacactcagagagacttatggggaaatggctgactgttgcgcaaaacaggagcctgaacggaatgagtgtttcctccagcacaaggatgacaacccaaatctgccccgcctcgtgcgacctgaggtcgatgtgatgtgcaccgcctttcatgacaacgaagagacattcctgaagaaatacctgtatgaaattgctcgtaggcacccatacttttatgcccccgagctcctgttctttgcaaagagatacaaagctgccttcactgaatgttgccaggcagctgataaggccgcatgtctcctgcctaaactggacgagctccgggatgaaggtaaggcttccagcgccaaacagcgcctgaagtgcgcttctctccagaagtttggcgagcgagcattcaaagcctgggctgtggcccgtctcagtcagaggtttccaaaggcagaatttgctgaggtctcaaaactggtgaccgacctcacaaaggtccatactgagtgttgccacggagatctgctggaat SEQ ID NO: 67QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARADVGYCTDRTCAKAPAWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGAASDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCAvSLQKFGERAFKAWAVARLvSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG SEQ ID NO: 68QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGAASDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARADVGYCTDRTCAKAPAWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGB2B3-1 (H3-mHSA-B1D2)

1

51

101

151

201

251

DAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQSPFEDH VKLVNEVTEF 301AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN 351ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF 401YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK 451CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD 501LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP 551ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL 601AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG 651EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA 701EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP 751KEFQAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD 801DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGL

 QVQLVQSGAE 851

901

951

1001

1051

CDR loops are highlighted within H3 (underlined 1-248 with bolditalicized CDRs) and B1D2 (underlined 841-1095 with bold italicizedCDRs). Connectors to modified HSA are dotted-underlined.Sequence Alignments for Various HSA Linker Conjugates Comprising B2B3and mHSA

1                                          45 A5-mHSA-ML3.9 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL A5-mHSA-B1D2 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL A5-mHSA-F5B6H2 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL B12-mHSA-B1D2 (1)QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGL B12-mHSA-F5B6H2 (1)QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGL F4-mHSA-B1D2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL F4-mHSA-F5B6H2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL H3-mHSA-B1D2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL H3-mHSA-F5B6H2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL46                                         90 A5-mHSA-ML3.9 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED A5-mHSA-B1D2 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED A5-mHSA-F5B6H2 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED B12-mHSA-B1D2 (46)EWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRPED B12-mHSA-F5B6H2 (46)EWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRPED F4-mHSA-B1D2 (46)EWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED F4-mHSA-F5B6H2 (46)EWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED H3-mHSA-B1D2 (46)EWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAED H3-mHSA-F5B6H2 (46)EWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAED91                                        135 A5-mHSA-ML3.9 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS A5-mHSA-B1D2 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS A5-mHSA-F5B6H2 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS B12-mHSA-B1D2 (91)TAVYYCARDLGAKQWLEGFDYWGQGTLVTVSSASTGGGGSGGGGS B12-mHSA-F5B6H2 (91)TAVYYCARDLGAKQWLEGFDYWGQGTLVTVSSASTGGGGSGGGGS F4-mHSA-B1D2 (91)TAVYYCAKGYSSSWSEVASGYWGQGTLVTVSSASTGGGGSGGGGS F4-mHSA-F5B6H2 (91)TAVYYCAKGYSSSWSEVASGYWGQGTLVTVSSASTGGGGSGGGGS H3-mHSA-B1D2 (91)TAVYYCARDR----GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS H3-mHSA-F5B6H2 (91)TAVYYCARDR----GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS136                                       180 A5-mHSA-ML3.9 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ A5-mHSA-B1D2 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ A5-mHSA-F5B6H2 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ B12-mHSA-B1D2 (136)GGGGSSYELTQDPA-VSVALGQTVRITCQGDSLRS---YYASWYQ B12-mHSA-F5B6H2 (136)GGGGSSYELTQDPA-VSVALGQTVRITCQGDSLRS---YYASWYQ F4-mHSA-B1D2 (136)GGGGSAIVMTQSPSSLSASVGDRVTITCRASQGIR---NDLGWYQ F4-mHSA-F5B6H2 (136)GGGGSAIVMTQSPSSLSASVGDRVTITCRASQGIR---NDLGWYQ H3-mHSA-B1D2 (132)GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ H3-mHSA-F5B6H2 (132)GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ181                                       225 A5-mHSA-ML3.9 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED A5-mHSA-B1D2 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED A5-mHSA-F5B6H2 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED B12-mHSA-B1D2 (177)QKPGQAPVLVIYGKNNRPSGIPDRFSGSTSGNSASLTITGAQAED B12-mHSA-F5B6H2 (177)QKPGQAPVLVIYGKNNRPSGIPDRFSGSTSGNSASLTITGAQAED F4-mHSA-B1D2 (178)QKAGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPDD F4-mHSA-F5B6H2 (178)QKAGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPDD H3-mHSA-B1D2 (176)QHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADD H3-mHSA-F5B6H2 (176)QHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADD226                                       270 A5-mHSA-ML3.9 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD A5-mHSA-B1D2 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD A5-mHSA-F5B6H2 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD B12-mHSA-B1D2 (222)EADYYCNSRDSSGNHWVFGGGTKVTVLG-AASDAHKSEVAHRFKD B12-mHSA-F5B6H2 (222)EADYYCNSRDSSGNHWVFGGGTKVTVLG-AASDAHKSEVAHRFKD F4-mHSA-B1D2 (223)FATYFCQQAHSF--PPTFGGGTKVEIKRGAASDAHKSEVAHRFKD F4-mHSA-F5B6H2 (223)FATYFCQQAHSF--PPTFGGGTKVEIKRGAASDAHKSEVAHRFKD H3-mHSA-B1D2 (221)EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD H3-mHSA-F5B6H2 (221)EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD271                                       315 A5-mHSA-ML3.9 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES A5-mHSA-B1D2 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES A5-mHSA-F5B6H2 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES B12-mHSA-B1D2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES B12-mHSA-F5B6H2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES F4-mHSA-B1D2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES F4-mHSA-F5B6H2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES H3-mHSA-B1D2 (265)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES H3-mHSA-F5B6H2 (265)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES316                                       360 A5-mHSA-ML3.9 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL A5-mHSA-B1D2 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL A5-mHSA-F5B6H2 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL B12-mHSA-B1D2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL B12-mHSA-F5B6H2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL F4-mHSA-B1D2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL F4-mHSA-F5B6H2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL H3-mHSA-B1D2 (310)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL H3-mHSA-F5B6H2 (310)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL361                                       405 A5-mHSA-ML3.9 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY A5-mHSA-B1D2 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY A5-mHSA-F5B6H2 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY B12-mHSA-B1D2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY B12-mHSA-F5B6H2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY F4-mHSA-B1D2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY F4-mHSA-F5B6H2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY H3-mHSA-B1D2 (355)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY H3-mHSA-F5B6H2 (355)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY406                                       450 A5-mHSA-ML3.9 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS A5-mHSA-B1D2 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS A5-mHSA-F5B6H2 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS B12-mHSA-B1D2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS B12-mHSA-F5B6H2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS F4-mHSA-B1D2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS F4-mHSA-F5B6H2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS H3-mHSA-B1D2 (400)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS H3-mHSA-F5B6H2 (400)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS451                                       495 A5-mHSA-ML3.9 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL A5-mHSA-B1D2 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL A5-mHSA-F5B6H2 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL B12-mHSA-B1D2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL B12-mHSA-F5B6H2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL F4-mHSA-B1D2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL F4-mHSA-F5B6H2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL H3-mHSA-B1D2 (445)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL H3-mHSA-F5B6H2 (445)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL496                                       540 A5-mHSA-ML3.9 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL A5-mHSA-B1D2 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL A5-mHSA-F5B6H2 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL B12-mHSA-B1D2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL B12-mHSA-F5B6H2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL F4-mHSA-B1D2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL F4-mHSA-F5B6H2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL H3-mHSA-B1D2 (490)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL H3-mHSA-F5B6H2 (490)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL541                                       585 A5-mHSA-ML3.9 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG A5-mHSA-B1D2 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG A5-mHSA-F5B6H2 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG B12-mHSA-B1D2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG B12-mHSA-F5B6H2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG F4-mHSA-B1D2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG F4-mHSA-F5B6H2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG H3-mHSA-B1D2 (535)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG H3-mHSA-F5B6H2 (535)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG586                                       630 A5-mHSA-ML3.9 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV A5-mHSA-B1D2 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV A5-mHSA-F5B6H2 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV B12-mHSA-B1D2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV B12-mHSA-F5B6H2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV F4-mHSA-B1D2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV F4-mHSA-F5B6H2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV H3-mHSA-B1D2 (580)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV H3-mHSA-F5B6H2 (580)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV631                                       675 A5-mHSA-ML3.9 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV A5-mHSA-B1D2 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV A5-mHSA-F5B6H2 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV B12-mHSA-B1D2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV B12-mHSA-F5B6H2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV F4-mHSA-B1D2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV F4-mHSA-F5B6H2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV H3-mHSA-B1D2 (625)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV H3-mHSA-F5B6H2 (625)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV676                                       720 A5-mHSA-ML3.9 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL A5-mHSA-B1D2 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL A5-mHSA-F5B6H2 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL B12-mHSA-B1D2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL B12-mHSA-F5B6H2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL F4-mHSA-B1D2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL F4-mHSA-F5B6H2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL H3-mHSA-B1D2 (670)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL H3-mHSA-F5B6H2 (670)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL721                                       765 A5-mHSA-ML3.9 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT A5-mHSA-B1D2 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT A5-mHSA-F5B6H2 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT B12-mHSA-B1D2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT B12-mHSA-F5B6H2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT F4-mHSA-B1D2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT F4-mHSA-F5B6H2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT H3-mHSA-B1D2 (715)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT H3-mHSA-F5B6H2 (715)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT766                                       810 A5-mHSA-ML3.9 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA A5-mHSA-B1D2 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA A5-mHSA-F5B6H2 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA B12-mHSA-B1D2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA B12-mHSA-F5B6H2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA F4-mHSA-B1D2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA F4-mHSA-F5B6H2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA H3-mHSA-B1D2 (760)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA H3-mHSA-F5B6H2 (760)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA811                                       855 A5-mHSA-ML3.9 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA A5-mHSA-B1D2 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA A5-mHSA-F5B6H2 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG B12-mHSA-B1D2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA B12-mHSA-F5B6H2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG F4-mHSA-B1D2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA F4-mHSA-F5B6H2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG H3-mHSA-B1D2 (805)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA H3-mHSA-F5B6H2 (805)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG856                                       900 A5-mHSA-ML3.9 (847)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG A5-mHSA-B1D2 (847)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG A5-mHSA-F5B6H2 (847)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR B12-mHSA-B1D2 (851)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG B12-mHSA-F5B6H2 (851)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR F4-mHSA-B1D2 (851)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG F4-mHSA-F5B6H2 (851)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR H3-mHSA-B1D2 (850)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG H3-mHSA-F5B6H2 (850)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR901                                       945 A5-mHSA-ML3.9 (892)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH A5-mHSA-B1D2 (892)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH A5-mHSA-F5B6H2 (892)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM B12-mHSA-B1D2 (896)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH B12-mHSA-F5B6H2 (896)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM F4-mHSA-B1D2 (896)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH F4-mHSA-F5B6H2 (896)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM H3-mHSA-B1D2 (895)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH H3-mHSA-F5B6H2 (895)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM946                                       990 A5-mHSA-ML3.9 (937)DVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSSGGGSGGGGS A5-mHSA-B1D2 (937)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS A5-mHSA-F5B6H2 (937)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG B12-mHSA-B1D2 (941)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS B12-mHSA-F5B6H2 (941)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG F4-mHSA-B1D2 (941)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS F4-mHSA-F5B6H2 (941)TS----------NAVGFDYWGQGTLVTVSSGGGGSGGGSGGGGSG H3-mHSA-B1D2 (940)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS H3-mHSA-F5B6H2 (940)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG   991                                      1035 A5-mHSA-ML3.9 (982)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA A5-mHSA-B1D2 (982)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA A5-mHSA-F5B6H2 (972)QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA B12-mHSA-B1D2 (986)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA B12-mHSA-F5B6H2 (976)QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA F4-mHSA-B1D2 (986)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA F4-mHSA-F5B6H2 (976)QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA H3-mHSA-B1D2 (985)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA H3-mHSA-F5B6H2 (975)QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA1036                                     1080 A5-mHSA-ML3.9 (1026)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC A5-mHSA-B1D2 (1026)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC A5-mHSA-F5B6H2 (1017)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC B12-mHSA-B1D2 (1030)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC B12-mHSA-F5B6H2 (1021)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC F4-mHSA-B1D2 (1030)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC F4-mHSA-F5B6H2 (1021)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC H3-mHSA-B1D2 (1029)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC H3-mHSA-F5B6H2 (1020)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC 1081                1104A5-mHSA-ML3.9 (1071) ASWDYTLSGWVFGGGTKLTVLG-- A5-mHSA-B1D2 (1071)ASWDYTLSGWVFGGGTKLTVLG-- A5-mHSA-F5B6H2 (1062) QSYDRRTPGWVFGGGTKLTVLG--B12-mHSA-B1D2 (1075) ASWDYTLSGWVFGGGTKLTVLG--

APPENDIX 2 ANTICANCER AGENTS Anticancer agents for combination withB2B3-1 Manufacturer/Proprietor Brand Name(s) Anti-IGF1R Antibodies AMG479 (fully humanized mAb) Amgen IMCA12 (fully humanized mAb) ImCloneNSC-742460 Dyax 19D12 (fully humanized mAb) CP751-871 (fully humanizedmAb) Pfizer H7C10 (humanized mAb) alphaIR3 (mouse) scFV/FC (mouse/humanchimera) EM/164 (mouse) MK-0646, F50035 Pierre Fabre Medicament, MerckSmall Molecules Targeting IGF1R NVP-AEW541 Novartis BMS-536,924(1H-benzoimidazol-2-yl)- Bristol-Myers Squibb 1H-pyridin-2-one)BMS-554,417 Bristol-Myers Squibb Cycloligan TAE226 PQ401 Anti-EGFRMonoclonal Antibodies INCB7839 Incyte Bevacizumab Avastin ® GenentechCetuximab Erbitux ® IMCLONE mAb 806 Matuzumab (EMD72000) Nimotuzumab(TheraCIM) Panitumumab Vectibix ® Amgen Anti-ErbB3 Therapeutics — —U3-1287/AMG888 U3 Pharma/Amgen MM-121 Merrimack PharmaceuticalsAnti-ErbB2 Therapeutics — — trastuzumab Herceptin ® Genentech HKI-272 -neratinib Wyeth KOS-953 - tanespimycin Kosan Biosciences Her/ErbBDimerization Inhibitors 2C4, R1273 - Pertuzumab

 Omnitarg ® Genentech, Roche Small Molecules Targeting EGFR CI-1033 (PD183805) Pzifer, Inc. EKB-569 Gefitinib IRESSA ™ AstraZeneca Lapatinib(GW572016) GlaxoSmithKline Lapatinib Ditosylate Tykerb ® SmithKlineBeecham Erlotinib HCl (OSI-774) Tarceva ® OSI Pharms PD158780 PKI-166Novartis Tyrphostin AG 1478 (4-(3-Chloroanillino)-6,7-dimethoxyquinazoline) Anti-cmet Antibody Therapies AVEO (AV299) AVEOAMG102 Amgen 5D5 (OA-5D5) Genentech Small Molecules Targeting cmetPHA665752 ARQ-650RP ArQule ARQ 197 ArQule Alkylating Agents BCNU→1,3-bis t2-chloroethyl)- nitrosourea Bendamustine Busulfan MyleranGlaxoSmithKline Carboplatin Paraplatin Bristol-Myers Squibb CarboquoneCarmustine CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl- 1-nitrosourea (methylCCNU) Chlorambucil Leukeran ® Smithkline Beecham Chlormethine Cisplatin(Cisplatinum, CDDP) Platinol Bristol-Myers Cyclophosphamide CytoxanBristol-Myers Squibb Neosar Teva Parenteral Dacarbazine (DTIC)Fotemustine Hexamethylmelamine (Altretamine, HMM) Hexalen ® MGI Pharma,Inc. Ifosfamide Mitoxana ® ASTA Medica Lomustine Mannosulfan MelphalanAlkeran ® GlaxoSmithKline Nedaplatin Nimustine Oxaliplatin Eloxatin ®Sanofi-Aventis US Prednimustine, Matulane Sigma-Tau Pharmaceuticals,Inc. Procarbazine HCL Ribonucleotide Reductase Inhibitor (RNR)Ranimustine Satraplatin Semustine Streptozocin Temozolomide TreosulfanTriaziquone Triethylene Melamine ThioTEPA Bedford, Abraxis, TevaTriplatin tetranitrate Trofosfamide Uramustine Antimetabolites5-azacytidine Flourouracil (5-FU)/Capecitabine 6-mercaptopurine(Mercaptopurine, 6-MP) 6-Thioguanine (6-TG) Purinethol ® Teva CytosineArabinoside (Cytarabine, Thioguanine ® GlaxoSmithKline Ara-C)Azathioprine Azasan ® AAIPHARMA LLC Capecitabine XELODA ® HLR (Roche)Cladribine (2-CdA, 2- Leustatin ® Ortho Biotech chlorodeoxyadenosine)5-Trifluoromethyl-2′-deoxyuridine Fludarabine phosphate Fludara ® BayerHealth Care Floxuridine (5-fluoro-2) FUDR ® Hospira, Inc. Methotrexatesodium Trexall Barr Pemetrexed Alimta ® Lilly Pentostatin Nipent ®Hospira, Inc. Raltitrexed Tomudex ® AstraZeneca Tegafur AromatoseInhibitor Ketoconazole Glucocorticoids Dexamethasone Decadron ®Dexasone, Wyeth, Inc. Diodex, Hexadrol, Maxidex Prednisolone PrednisoneDeltasone, Orasone, Liquid Pred, Sterapred ® Immunotherapeutics Alphainterferon Angiogenesis Inhibitor Avastin ® Genentech IL-12→ Interleukin12 IL-2→ Interleukin 2 (Aldesleukin) Proleukin ® Chiron KinaseInhibitors AMG 386 Amgen Axitinib ((AG-013736) Pfizer, Inc Bosutinib(SKI-606) Wyeth Brivanib alalinate (BMS-582664) BMS Cediranib (AZD2171)Recentin AstraVeneca Dasatinib (BMS-354825) Sprycel ® Bristol-MyersSquibb Imatinib mesylate Gleevec Novartis Lestaurtinib (CEP-701)Cephalon Motesanib diphosphate (AMG-706) Amgen/Takeda Nilotinibhydrochloride monohydrate Tasigna ® Novartis Pazopanib HCL (GW786034)Armala GSK Semaxanib (SU5416) Pharmacia, Sorafenib tosylate Nexavar ®Bayer Sunitinib malate Sutent ® Pfizer, Inc. Vandetanib (AZD647) ZactimaAstraZeneca Vatalanib; PTK-787 Novartis; Bayer Schering Pharma XL184,NSC718781 Exelixis, GSK Microtubule-Targeting Agents ColchicineDocetaxel Taxotere ® Sanofi-Aventis US Ixabepilone IXEMPRA ™Bristol-Myers Squibb Larotaxel Sanofi-aventis Ortataxel SpectrumPharmaceuticals Nanoparticle paclitaxel (ABI-007) Abraxane ® AbraxisBioScience, Inc. Paclitaxel Taxol ® Bristol-Myers Squibb Tesetaxel GentaVinblastine sulfate Velban ® Lilly Vincristine Oncovin ® Lilly Vindesinesulphate Eldisine ® Lilly Vinflunine Pierre Fabre Vinorelbine tartrateNavelbine ® Pierre Fabre mTOR Inhibitors Deforolimus (AP23573, MK 8669)ARIAD Pharmaceuticals, Inc Everolimus (RAD001, RAD001C) Certican ®,Afinitor Novartis Sirolimus (Rapamycin) Rapamune ® Wyeth PharamaTemsirolimus (CCI-779) Torisel ® Wyeth Pharama Protein SynthesisInhibitor L-asparaginase Elspar ® Merck & Co. Somatostatin AnalogueOctreotide acetate Sandostatin ® Novartis Topoisomerase InhibitorsActinomycin D Camptothecin (CPT) Belotecan Daunorubicin citrateDaunoxome ® Gilead Doxorubicin hydrochloride Doxil ® Alza Vepesid ®Bristol-Myers Squibb Etoposide Etopophos Hospira, Bedford, TevaParenteral, Etc. Irinotecan HCL (CPT-11) Camptosar ® Pharmacia & UpjohnMitoxantrone HCL Novantrone EMD Serono Rubitecan Teniposide (VM-26)Vumon ® Bristol-Myers Squibb Topotecan HCL Hycamtin ® GlaxoSmithKlineChemotherapeutic Agents Adriamycin, 5-Fluorouracil, Cytoxin, Bleomycin,Mitomycin C, Daunomycin, Carminomycin, Aminopterin, Dactinomycin,Mitomycins, Esperamicins Clofarabine, Mercaptopurine, Pentostatin,Thioguanine, Cytarabine, Decitabine, Floxuridine, Gemcitabine (Gemzar),Enocitabine, Sapacitabine Hormonal Therapies Abarelix Plenaxis ™ AmgenAbiraterone acetate CB7630 BTG plc Afimoxifene TamoGel AscendTherapeutics, Inc. Anastrazole Arimidex ® AstraZeneca Aromataseinhibitor Atamestane plus toremifene Intarcia Therapeutics, Inc.Arzoxifene Eli Lilly & Co. Asentar; DN 101 Novartis; Oregon Health &Science Univ. Bicalutamide Casodex ® AstraZeneca Buserelin Suprefact ®Sanofi Aventis Cetrorelix Cetrotide ® EMD Serono Exemestane Aromasin ®Pfizer Exemestane Xtane Natco Pharma, Ltd. Fadrozole (CGS 16949A)Flutamide Eulexin ® Schering Flutamide Prostacur Laboratorios Almirall,S.A. Fulvestrant Faslodex ® AstraZeneca Goserelin acetate Zoladex ®AstraZeneca Letrozole Femara ® Novartis Letrozole (CGS20267) FemaraChugai Pharmaceutical Co., Ltd. Letrozole Estrochek JagsonpalPharmaceuticals, Ltd. Letrozole Letrozole Indchemie Health SpecialitiesLeuprolide acetate Eligard ® Sanofi Aventis Leuprolide acetate LeoprilVHB Life Sciences, Inc. Leuprolide acetate Lupron ®/Lupron Depot TAPPharma Leuprolide acetate Viador Bayer AG Megestrol acetate Megace ®Bristol-Myers Squibb Magestrol acetate Estradiol Valerate JagsonpalPharmaceuticals, Ltd. (Delestrogen) Medroxyprogesterone acetate VeraplexCombiphar MT206 Medisyn Technologies, Inc. Nafarelin Nandrolonedecanoate Zestabolin Mankind Pharma, Ltd. Nilutamide Nilandron ® AventisPharmaceuticals Raloxifene HCL Evista ® Lilly Tamoxifen Taxifen YungShin Pharmaceutical Tamoxifen Tomifen Alkem Laboratories, Ltd. Tamoxifencitrate Nolvadex AstraZeneca Tamoxifen citrate Soltamox EUSA Pharma,Inc. Tamoxifen citrate Tamoxifen citrate Sopharma JSCo. SOPHARMAToremifene citrate Fareston ® GTX, Inc. Triptorelin pamoate Trelstar ®Watson Labs Triptorelin pamoate Trelstar Depot Paladin Labs, Inc.Protein Kinase B (PKB) Inhibitors Akt Inhibitor ASTEX Astex TherapeuticsAkt Inhibitors NERVIANO Nerviano Medical Sciences AKT Kinase InhibitorTELIK Telik, Inc. AKT DECIPHERA Deciphera Pharmaceuticals, LLCPerifosine (KRX0401, D-21266) Keryx Biopharmaceuticals, Inc., AEternaZentaris, Inc. Perifosine with Docetaxel Keryx Biopharmaceuticals, Inc.,AEterna Zentaris, Inc. Perifosine with Gemcitabine AEterna Zentaris,Inc. Perifosine with Paclitaxel Keryx Biopharmaceuticals, Inc, AEternaZentaris, Inc. Protein Kinase-B inhibitor DEVELOGEN DeveloGen AG PX316Oncothyreon, Inc. RX0183 Rexahn Pharmaceuticals, Inc. RX0201 RexahnPharmaceuticals, Inc. VQD002 VioQuest Pharmaceuticals, Inc. XL418Exelixis, Inc. ZEN027 AEterna Zentaris, Inc. Phosphatidylinositol3-Kinase (PI3K) Inhibitors BEZ235 Novartis AG BGT226 Novartis AG CAL101Calistoga Pharmaceuticals, Inc. CHR4432 Chroma Therapeutics, Ltd.Erk/PI3K Inhibitors ETERNA AEterna Zentaris, Inc. GDC0941 GenentechInc./Piramed Limited/Roche Holdings, Ltd. Enzastaurin HCL (LY317615)Enzastaurin Eli Lilly LY294002/Wortmannin PI3K Inhibitors SEMAFORESemafore Pharmaceuticals PX866 Oncothyreon, Inc. SF1126 SemaforePharmaceuticals VMD-8000 VM Discovery, Inc. XL147 Exelixis, Inc. XL147with XL647 Exelixis, Inc. XL765 Exelixis, Inc. PI-103 Roche/PiramedCyclin-dependent kinase inhibitors CYC200, r-roscovitine SeliciclibCyclacel Pharma NSC-649890, L86-8275, HMR-1275 Alvocidib NCI TLr9, CD289IMOxine Merck KGaA HYB2055 Idera IMO-2055 Isis Pharma 1018 ISS DynavaxTechnologies/UCSF PF-3512676 Pfizer Enzyme Inhibitor Lonafarnib(SCH66336) Sarasar SuperGen, U Arizona Anti-TRAIL AMG-655 AeternaZentaris, Keryx Biopharma Apo2L/TRAIL, AMG951 Genentech, Amgen Apomab(fully humanized mAb Genentech Target Other Imprime PGG BiotheraCHR-2797 AminopeptidaseM1 Chroma Therapeutics E7820, NSC 719239Integrin-alpha2 Eisai INCB007839 ADAM 17, TACE Incyte CNF2024, BIIB021Hsp90 Biogen Idec MP470, HPK-56 Kit/Met/Ret Shering-PloughSNDX-275/MS-275 HDAC Syndax Zarnestra, Tipifarnib, R115777 Ras JanssenPharma Volociximab; Eos 200-4, M200 alpha581 integrin Biogen Idec; EliLilly/UCSF/PDL BioPharma Apricoxib (TP2001) COX-2 Inhibitor DaiichiSankyo; Tragara Pharma

indicates data missing or illegible when filed

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All patents and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if eachindependent patent or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

1.-36. (canceled)
 37. A conjugate comprising a human serum albumin (HSA)linker having an amino terminus and a carboxy terminus, said HSA linkercomprising an amino acid sequence having at least 95% sequence identityto the sequence set forth in SEQ ID NO: 1 and comprising a first bindingmoiety bonded to the amino terminus of said HSA linker and a secondbinding moiety bonded to the carboxy terminus of said HSA linker,wherein said first binding moiety specifically binds ErbB3 with adissociation constant (K_(d)) of about 16 nM, as determined by surfaceplasmon resonance, and said second binding moiety specifically bindsErbB2 with a K_(d) of about 0.3 nM, as determined by surface plasmonresonance.
 38. The conjugate of claim 37, wherein the HSA linker furthercomprises a peptide connector comprising 2 to 20 amino acid residues atthe amino or carboxy terminus of said HSA linker, wherein said peptideconnector covalently joins said HSA linker to said first or secondbinding moiety.
 39. A composition comprising the conjugate of claim 37admixed with a pharmaceutically acceptable carrier, excipient, ordiluent.
 40. The composition of claim 39 in a solution comprising 25mg/mL of the conjugate.
 41. The conjugate of claim 37, wherein saidfirst binding moiety is a single chain variable fragment (scFv)comprising the amino acid sequence of SEQ ID NO: 26 and said secondbinding moiety is an scFv comprising the amino acid sequence of SEQ IDNO:
 27. 42. The conjugate of claim 38, wherein said HSA linker comprisesa peptide connector of 2 to 20 amino acid residues between said firstbinding moiety and said HSA linker and a peptide connector of 2 to 20amino acid residues between said second binding moiety and said HSAlinker.
 43. The conjugate of claim 37, wherein said HSA linker comprisesa serine residue at position 34 of SEQ ID NO: 1 and a glutamine residueat position 503 of SEQ ID NO:
 1. 44. The conjugate of claim 37, whereinsaid HSA linker comprises the amino acid sequence of SEQ ID NO:
 1. 45.The conjugate of claim 37, wherein each of said first and second bindingmoieties is an scFv.
 46. The conjugate of claim 45, wherein each saidscFv is human or humanized.
 47. The conjugate of claim 37, wherein saidconjugate exhibits an in vivo half-life of greater than 8 hours.
 48. Theconjugate of claim 37, wherein said HSA linker comprises amino acidresidues 25-44 and amino acid residues 494-513 of SEQ ID NO:1.
 49. Theconjugate of claim 48, wherein said HSA linker comprises amino acidresidues 25-70 and amino acid residues 450-513 of SEQ ID NO:1.
 50. Theconjugate of claim 49, wherein said HSA linker comprises amino acidresidues 15-100 and amino acid residues 400-520 of SEQ ID NO:1.
 51. Theconjugate of claim 50, wherein said HSA linker comprises amino acidresidues 10-200 and amino acid residues 300-575 of SEQ ID NO:1.
 52. Theconjugate of claim 51, wherein said HSA linker comprises amino acidresidues 5-250 and amino acid residues 275-580 of SEQ ID NO:1.
 53. Theconjugate of claim 37, wherein said conjugate exhibits the ability tobind both ErbB2 and ErbB3 before and after incubation in human serum at37° C. for 120 hours as measured by enzyme-linked immunosorbent assay(ELISA).
 54. A kit comprising the conjugate of claim 37 in a packagewith instructions for administering said conjugate to a patient.